WO2018149929A1 - Combination of reporter gene assays and transcriptional analysis - Google Patents

Combination of reporter gene assays and transcriptional analysis Download PDF

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WO2018149929A1
WO2018149929A1 PCT/EP2018/053810 EP2018053810W WO2018149929A1 WO 2018149929 A1 WO2018149929 A1 WO 2018149929A1 EP 2018053810 W EP2018053810 W EP 2018053810W WO 2018149929 A1 WO2018149929 A1 WO 2018149929A1
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cells
cell
based assay
qpcr
probe
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French (fr)
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Stefan Golz
Svenja KLEIMANN
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Bayer Pharma Aktiengesellschaft
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
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    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0045—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent agent being a peptide or protein used for imaging or diagnosis in vivo
    • A61K49/0047—Green fluorescent protein [GFP]
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to the field of molecular and cell biology, more particularly, the present invention relates to a method which combines two or more techniques to analyse the effects of compounds on cells or tissues.
  • the invention also relates to a method which combines methods for the analysis or quantification of RNA with methods to measure second messenger signaling or promotor activity.
  • HTS is performed, for example, in 1536-well plates with assay volumes between 5-10 ⁇ . This set up, together with fully-automated robotic systems allows for testing in excess of 200,000 compounds per day. Comprehensive substance collections together with sophisticated screening technologies have resulted in a clear advantage in lead discovery especially for poorly draggable targets. The productivity of HTS has recently been questioned, because often no modulators could be found for a given target, and the poor drugability of many molecular targets pursued in research programs, together with a poor quality of compounds in screening libraries, have been identified as major drawbacks responsible for low success. Improvements in the design of library screening methods aim at the identification of compounds with more "drug-like" physico-chemical properties, like oral availability. Summary of the invention
  • embodiments disclosed herein are not meant to be understood as individual embodiments which would not relate to one another.
  • Features discussed with one embodiment are meant to be disclosed also in connection with other embodiments shown herein. If, in one case, a specific feature is not disclosed with one embodiment, but with another, the skilled person would understand that does not necessarily mean that said feature is not meant to be disclosed with said other embodiment. The skilled person would understand that it is the gist of this application to disclose said feature also for the other embodiment, but that just for purposes of clarity and to keep the specification in a manageable volume this has not been done.
  • the content of the prior art documents referred to herein is incorporated by reference. This refers, particularly, to prior art documents that disclose standard or routine methods. In that case, the incorporation by reference has mainly the purpose to provide sufficient enabling disclosure, and avoid lengthy repetitions.
  • a method for analysing the effects of one or more test compounds on cells or tissues comprising at least
  • ( ⁇ ) a step comprising an expression analysis related to said cells or tissues.
  • the combination of a cell-based assay and an expression analysis readout has a number of unexpected advantages, namely, saving of time, saving of resources, enhanced assay accuracy, and combination of data from different pathway levels.
  • cell-based assay relates to methods using defined cells, which are then exposed the test compounds.
  • Biochemical assays are target-based in vitro assays which historically have been the mainstay of high throughput screening (HTS) in the pharmaceutical industry. Such assays include assessment of enzymatic activity (e.g. for kinases, proteases, or transferases), receptor-ligand binding (e.g., for G-protein coupled receptors (GPCRs), ion channels , or nuclear receptors), or protein-protein interactions. Biochemical assays are often direct and specific to the target of interest and can be miniaturized readily. However, not all targets can be purified or prepared in a manner suitable for biochemical measurement. Additionally, a certain activity of a small molecule observed in an in vitro assay does not always translate into the same activity in a cellular context, because of issues including membrane permeability, off-target effects, and cytotoxicity [Tolliday, 2009].
  • cell-based assays do not require an a priori knowledge of the target that might be affected by the test compounds. Further, in contrast to biochemical assays, the likelihood that a extrapolation from assay results into a living system can be done is much higher. In most cell based assays, entire pathways of interest can be interrogated, providing the opportunity for multiple potential intervention points, as opposed to a single predefined step with the biochemical approach. Examples for cell based assays formats are (but not limited to) proliferation assays, reporter gene assays, second messenger assays and high content screening assays. In such way, parameters such as potential drug cytotoxicity, mechanism of action, and biological activity can be determined.
  • expression analysis relates to a method involving the qualitative or quantitative determination of the expression of one gene, or a set of gene, either measured on the protein level or on the mRNA level.
  • the cell-based assay format is based on measuring second messenger signaling or promotor activity [Roda, 2010].
  • said cell- based assay format is a reporter gene based assay format.
  • cells can be engineered to express specific gene products in response to a given stimulus.
  • the gene product itself may possess an inherent property that enables it to be measured directly, e.g., green fluorescent protein (GFP), or it may display enzymatic activity that can be monitored, e.g., luciferase.
  • the gene product may respond to changes in the levels of a signaling molecule, e.g., the Ca 2+ ion-mediated activation of aequorin luminescence.
  • GFP green fluorescent protein
  • the gene product may respond to changes in the levels of a signaling molecule, e.g., the Ca 2+ ion-mediated activation of aequorin luminescence.
  • the choice of a particular promoter, the number of promoter copies per reporter gene unit, and the nature of the reporter gene allow to control the basal level of reporter gene activityand control of the degree of stimulation measured.
  • Endogenous promoters such as c-fos, the cAMP response element (CRE), or the estrogen response element are commonly used.
  • CRE cAMP response element
  • the accuracy of assays employing these promoters istheir activation through endogenous intracellular signaling events.
  • the reporter gene itself should ultimately generate a signal that can be clearly identified.
  • the reporter gene products can be either intracellular or extracellular in nature. Intracellular products are retained in the cell for quantification in situ or following cell lysis.
  • Extracellular products are secreted into the extracellular medium for assay, allowing repeated experimentation and sampling without disrupting the cells.
  • Commonly used intracellular reporter genes are chloramphenicol acetyltransferase (CAT), ⁇ -galactosidase, luciferase, aequorin, and GFP.
  • Extracellular reporter genes are usually secreted placental alkaline phosphatase (SPAP) or ⁇ - lactamase [Tolliday, 2009].
  • a reporter gene is chosen the gene product of which can easily be detected with standard biochemical or histochemical methods.
  • Two commonly used types of reporter genes are resistence genes and reporter genes.
  • Resistance genes are genes, the expression of which confers on a cell the resistance to antibiotics or other substances which would, in the absence of the resistence gene, cause the death of the cell.
  • reporter genes or proteins can be fused to a target genes to detect expression thereof by luminescent or fluorescent readouts (e.g. GFP, luciferase), namely because the reporter gene is coexpressed with the target gene.
  • luminescent or fluorescent readouts e.g. GFP, luciferase
  • said cell- based assay format is at least one selected from the group consisting of:
  • Luminescence is the term given to the emission of photons in the visible spectral range, with this emission being brought about by excitated emitter molecules. In contrast to fluorescence, the energy for this is not supplied externally in the form of radiation of shorter wavelength.
  • chemiluminescence is the term given to a chemical reaction which leads to an excited molecule which itself emits light when the excited electrons return to the normal energy level.
  • Bioluminescence is the term used when this reaction is catalyzed by an enzyme.
  • the enzymes which participate in the reaction are generally termed luciferases. Luciferases are peroxidases or monooxygenases and dioxygenases.
  • the enzyme substrates, which form the starting substances for the light- emitting products, are termed luciferins. They differ from species to species.
  • the quantum yield of the systems lies between 0.1 and 0.9 photons per transformed substrate molecule.
  • Luciferases can be classified on the basis of their origin or their enzymic properties. Luciferases can also be distinguished from each other on the basis of their substrate specificity. The most important substrates include coelenterazine and luciferin, and also derivatives of the two substances. An overview, but not limited to, of some luciferases:
  • Renilla Luciferase (Renilla reniformis) - substrate Coelenterazine
  • Watasemia Luciferase (Watasenia scintillans) - substrate Watasemia Luciferin
  • Olophorus Luciferase (Olophorus gracilirostris) - substrate Coelenterazine
  • Firefly Luciferase (Photinus pyralis) - substrate: Firefly Luciferin
  • Hydozoa (aequoria, halistaura obelia) and anthropods (acanthotilum, sea cactus, cavernularia, renila, ptilosarcus, stylatula).
  • ⁇ Green fluorescent protein (Aequorea macrodactyla, Gene ID: AF435433)
  • Green fluorescent protein Heteractis crispa, Gene ID: AF420592
  • Green fluorescent protein-like protein (Montastraea annularis, Gene ID: AY037766)
  • Green fluorescent protein-like protein (Montastraea cavernosa, Gene ID: AY037768)
  • Cyan fluorescent protein (Montastraea cavernosa, Gene ID: AY056460)
  • Green fluorescent protein (Renilla muelleri, Gene ID: AY015996)
  • Green fluorescent protein (Renilla renoformis, Gene ID: AF372525)
  • Green fluorescent protein- like protein (Ricordea florida, Gene ID: AY037774)
  • the fluorescent proteins differ from one another not only due to their nucleotide and amino acid sequences but also due to their biochemical and physical properties.
  • the spectral characteristics of the fluorescent proteins may differ both on the side of excitation and on the side of emission.
  • Fluorescence-based assays can be generally divided into two classes.
  • the first class encompasses techniques that macroscopically detect the total fluorescence intensity, fluorescence polarization, fluorescence resonance energy transfer (FRET), fluorescence lifetime, time-resolved fluorescence, and combinations of these techniques, such as time-resolved fluorescence polarization.
  • the second class of fluorescence-based assays detects fluorescence from single fluorescent molecules, such as fluorescence correlation spectroscopy and fluorescence intensity distribution analysis. These fluorescence techniques have been used to monitor an enormous collection of biological processes, such as macromolecule-macromolecule interactions, macromolecule-small molecule interactions, enzymatic activities, signal transduction, cell health, and states and locations of molecules, organelles, or cells [Tolliday, 2009] .
  • Fluorescence resonance energy transfer utilises non-radiative energy transfer from a donor fluorophore to an acceptor fluorophore resulting in fluorescence emission from the acceptor.
  • FRET can occur where there is sufficient overlap of the donor emission spectrum with the acceptor excitation spectrum, close proximity ( ⁇ 100 A) and correct donor-acceptor orientation of dipole moments. These parameters are readily adaptable to ligand binding assays, where binding of a fluorescent ligand with suitable spectral properties to a fluorophore-tagged protein provides the close proximity required for FRET.
  • FRET assays require the target protein to be tagged with a donor or acceptor fluorophore, this can be achieved by fusing the N-terminus of the protein with a fluorescent protein (such as EGFP) [Pfleger, 2015] .
  • a fluorescent protein such as EGFP
  • FA fluorescence anisotropy
  • the transcriptome is defined as the full complement of RNA transcripts of the genes of a cell or organism.
  • the types and relative abundance of different transcripts i.e. the messenger RNAs (mRNAs)
  • mRNAs messenger RNAs
  • This analysis is defined as transcriptomics.
  • Used methods for transcriptome analysis are, but not limited to, sequencing, microarrays and PCR-based methods (e.g. QPCR).
  • the expression analysis of said cells or tissues comprises an RNA quantification.
  • the expression analysis of said cells or tissues comprises a QPCR or RT-PCR.
  • RT-PCR means "real-time polymerase chain reaction” while the term QPCR means "quantitative polymerase chain reaction”
  • RT-PCR is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, i.e. in real-time, and not at its end, as in conventional PCR.
  • Real-time PCR can be used quantitatively (Quantitative real-time PCR), semi-quantitatively (Semi quantitative real-time PCR) or qualitatively (Qualitative real-time PCR).
  • Two common methods for the detection of PCR products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary sequence.
  • Real-time PCR is carried out in a thermal cycler with the capacity to illuminate each sample with a beam of light of at least one specified wavelength and detect the fluorescence emitted by the excited fluorophore.
  • the thermal cycler is also able to rapidly heat and chill samples, thereby taking advantage of the physicochemical properties of the nucleic acids and DNA polymerase.
  • the PCR process generally consists of a series of temperature changes that are repeated 25 - 50 times. These cycles normally consist of three stages: the first, at around 95 °C, allows the separation of the nucleic acid's double chain; the second, at a temperature of around 50-60 °C, allows the binding of the primers with the DNA template; [Rhoads, 1990] the third, at between 68 - 72 °C, facilitates the polymerization carried out by the DNA polymerase. Due to the small size of the fragments the last step is usually omitted in this type of PCR as the enzyme is able to increase their number during the change between the alignment stage and the denaturing stage.
  • the fluorescence is measured during short temperature phase lasting only a few seconds in each cycle, with a temperature of, for example, 80 °C, in order to reduce the signal caused by the presence of primer dimers when a non-specific dye is used.
  • the temperatures and the timings used for each cycle depend on a wide variety of parameters, such as: the enzyme used to synthesize the DNA, the concentration of divalent ions and deoxyribonucleotides (dNTPs) in the reaction and the bonding temperature of the primers.
  • Probes used in multiplex assays are conjugated with fluorescent reporter dyes, which vary in their absorption and emission spectra.
  • the number of genes that can be analyzed in the same tube is limited by spectral properties of the available fluorophores, which should have nonoverlapping emission spectra for separate detection.
  • the majority of modern spectrofluorometric thermal cyclers allow the measurement of fluorescence in four different wavelength channels (although six- channel machines do exist), thereby enabling the concurrent detection of up to four different DNA targets.
  • a choice of dyes optimized for detection in each channel is available for distinctive labelling of specific probes.
  • TaqMan is a technique in which the release of a fluorescent reporter dye from a hybridisation probe in real-time during a polymerase chain reaction (PCR) is proportional to the accumulation of the PCR product. Quantification is based on the early, linear part of the reaction, and by determining the threshold cycle (CT), at which fluorescence above background is first detected.
  • CT threshold cycle
  • the most widely used probes to monitor DNA amplification in real-time PCR in clinical settings are TaqMan or hydrolysis probes [Williams, 1996]. These probes are labeled at one end with a reporter fluorescent dye and on the other with a fluorescence quencher, which must exhibit spectral overlap with the fluorophore.
  • Quencher absorbs energy emitted by the flourophore through fluorescent resonance energy transfer (FRET); the quenchers, currently used in multiplex PCR, reemit energy as heat (dark quencher).
  • FRET fluorescent resonance energy transfer
  • the quenchers currently used in multiplex PCR, reemit energy as heat (dark quencher).
  • the Tm of the probe is usually 8-10°C higher than that of the primers, which allows the probe to anneal prior to extension.
  • the 5 -exonuclease activity of the Taq polymerase hydrolyses the bound probe and releases the dye from the quencher, relieving quenching effect. The level of detected fluorescence is therefore proportional to the amounts of newly synthesized DNA.
  • Hybridization probes consist of two single-dye labeled oligonucleotides which bind to adjacent targets in the amplified region, thereby bringing the two dyes in close proximity and inducing FRET.
  • Molecular beacon probes are designed as hairpins, with target sequence located in the loop and fluorescent dye and the quencher conjugated to the 5'-end and 3'-end of the oligonucleotide. In the intact, hairpin state, fluorescence is quenched; binding of the probe to the amplified sequence forces fluorophore and quencher apart, and releases fluorescent signal.
  • Scorpion primers Another widely used probes are Scorpion primers, which combine PCR primer and specific probe in one sequence [Little, 1999].
  • the structure of Scorpion primers promotes unimolecular probing mechanism; consequently, they can perform better than TaqMan probes or molecular beacons particularly under fast cycling conditions [Brown, 2000].
  • Described probes can be used not only to quantify levels of DNA, but to discriminate between alleles and detect mutations as well [Skoblov, 2011].
  • the cell-based assay format is a luciferase based assay format, and wherein said expression analysis of said cells or tissues is a QPCR or RT-PCR.
  • said cells are prokaryotic or eukaryotic cells.
  • said cells are selected from the group comprising human cells, animal cells, plant cells, microbial cells, and bacterial cells.
  • at least one of the steps is performed in at least one microtiter plate having a number of sample wells selected from the group consisting of 6, 24, 96, 384, 1536, 3072, 3456, 6144 and 9600 wells.
  • a microtiter plate or microplate or microwell plate or multiwell is a flat plate with multiple "wells" used as small test tubes.
  • the microplate has become a standard tool in analytical research and clinical diagnostic testing laboratories.
  • a microplate typically has, but not limited to, 6, 24, 96, 384, 1536, 3072, 6144 sample wells or even more arranged in a 2:3 rectangular matrix.
  • Some microplates have even been manufactured with 3456 or even 9600 wells, and an "array tape" product has been developed that provides a continuous strip of microplates embossed on a flexible plastic tape.
  • Each well of a microplate typically holds somewhere between tens of nanolitres to several millilitres of liquid. Wells can be either circular or square.
  • robots to specifically handle microplates. These robots may be liquid handlers which aspirate or dispense liquid samples from and to these plates, or "plate movers" which transport them between instruments, plate stackers which store microplates during these processes, plate hotels for longer term storage, plate washers for processing plates, plate thermal sealers for applying heat seals, de-sealers for removing heat seals, or microplate incubators to ensure constant temperature during testing. Instrument companies have designed plate readers which can detect specific biological, chemical or physical events in samples stored in these plates. Microtiter plates could be used for different cell-based assays formats, as well as expression analysis (e.g. QPCR).
  • QPCR expression analysis
  • microtiter plates allows the screening of libraries and or the integration into High Throughput Screening (HTS) enivironments, as discussed elsewhere herein.
  • HTS High Throughput Screening
  • a cell lysate or cell culture supernatant derived from the cell based assay step is subjected to the expression analysis step.
  • a method of screening a library of test compounds is provided, which method encompasses a method according to the above description.
  • library relates to a plurality of compounds of similar kind, e.g., a library of antibodies, a library of small molecules, or a library of aptamers. These libraries can have a size of anything between 5 to 10 15 individual compounds.
  • kits of parts comprising reagents suitable for performing the method according to the above description.
  • the use of such kit of parts is provided for identifying a particular gene or gene product to be a potential target for the action of potentially therapeutic agents.
  • the use of such kit of parts is provided for screening one or more test compounds which are potentially therapeutic agents, or a library thereof, for the development of a pharmaceutical drug.
  • the potentially therapeutic agents are preferably (i) biologies or (ii) small molecules.
  • biological refers to (i) protein-based moelcules, (ii) nucleic-acid an based molecules or (iii) cells or cell comprising entities, all of which have a physiological effect, preferably a therapeutic effect.
  • Molecules encompassed by that term are vaccines, cytokines, hormones, coagulation factors, antibodies and antibody-derived molecules, antibody mimetics, aptamers, RNA molecules and the like, and blood products and the like.
  • Cells or cell comprising entities encompassed by that term are stem cells and T cells, including transgenic or chimerized variants thereof, and cellular blood products and the like.
  • small molecule refers to a non-peptidic, non-oligomeric organic compound either synthesized in the laboratory or found in nature.
  • Small molecules can refer to compounds that are "natural product- like", however, the term “small molecule” is not limited to "natural product-like” compounds. Rather, a small molecule is typically characterized in that it contains several carbon-carbon bonds, and has a molecular weight of less than 2500, although this characterization is not intended to be limiting for the purposes of the present invention.
  • oligonucleotide as used herein is a stretch of nucleotide residues which has a sufficient number of bases to be used as an oligomer, amplimer or probe in a polymerase chain reaction (PCR). Oligonucleotides can be prepared from genomic or cDNA sequence and are used to amplify, reveal, or confirm the presence of a similar DNA or RNA in a particular cell or tissue. Oligonucleotides or oligomers comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 35 nucleotides, preferably about 25 nucleotides. "Probes" as used herein refers to nucleic acid probes.
  • nucleic acid probes may be used in southern, northern, PCR based methods, hybridisation based methods or in situ hybridizations to determine whether DNA or RNA encoding a certain protein is present in a cell type, tissue, organ or fluid.
  • Cells as used herein encompass prokaryotic and/or eukaryotic cells of a defined cell line, primary cells (a cell or cell line taken directly from a living organism, which is not immortalized), cells isolated from defined tissues or cell within a tissue from different species.
  • Primary cells a cell or cell line taken directly from a living organism, which is not immortalized
  • cells isolated from defined tissues or cell within a tissue from different species are examples of prokaryotic cells of a defined cell line, primary cells (a cell or cell line taken directly from a living organism, which is not immortalized), cells isolated from defined tissues or cell within a tissue from different species.
  • Primary cells a cell or cell line taken directly from a living organism, which is not immortalized
  • Prokaryotes refers to bacteria and archaea.
  • the labeled probe is selected so that its sequence is substantially complementary to a segment of the test locus or a reference locus. As indicated above, the nucleic acid site to which the probe binds should be located between the primer binding sites for the upstream and downstream amplification primers.
  • the primers used in the amplification are selected so as to be capable of hybridizing to sequences at flanking regions of the locus being amplified.
  • the primers are chosen to have at least substantial complementarity with the different strands of the nucleic acid being amplified.
  • the primers are selected in such that they flank the probe, i.e. are located upstream and downstream of the probe.
  • the primer must have sufficient length so that it is capable of priming the synthesis of extension products in the presence of an agent for polymerization.
  • the length and composition of the primer depends on many parameters, including, for example, the temperature at which the annealing reaction is conducted, proximity of the probe binding site to that of the primer, relative concentrations of the primer and probe and the particular nucleic acid composition of the probe.
  • the primer includes 15-30 nucleotides.
  • the length of the primer may be more or less depending on the complexity of the primer binding site and the factors listed above.
  • the labels used for labeling the probes or primers of the current invention and which can provide the signal corresponding to the quantity of amplification product can take a variety of forms.
  • a fluorescent signal is one signal which can be measured.
  • measurements may also be made, for example, by monitoring radioactivity, colorimetry, absorption, magnetic parameters, or enzymatic activity.
  • labels which can be employed include, but are not limited to, fluorophors, chromophores, radioactive isotopes, electron dense reagents, enzymes, and ligands having specific binding partners (e.g., biotin-avidin).
  • a number of labels useful for attachment to probes or primers are commercially available including fluorescein and various fluorescein derivatives such as, but not limited to FAM, HEX, TET and JOE; lucifer yellow, and coumarin derivatives.
  • Labels may be attached to the probe or primer using a variety of techniques and can be attached at the 5' end, and/or the 3' end and/or at an internal nucleotide.
  • the label can also be attached to spacer arms of various sizes which are attached to the probe or primer. These spacer arms are useful for obtaining a desired distance between multiple labels attached to the probe or primer.
  • a single label may be utilized; whereas, in other instances, such as with the 5' fluorogenic nuclease assays for example, two or more labels are attached to the probe.
  • the probe includes multiple labels, it is generally advisable to maintain spacing between the labels which is sufficient to permit separation of the labels during digestion of the probe through the 5'-3' nuclease activity of the nucleic acid polymerase.
  • fluorescent acceptor molecules like TAMRA
  • Quenchers are chemically related to fluorophores but instead of emitting absorbed fluorescence resonance energy as light they have the useful property of transforming the light energy to heat.
  • Heat dissipation of fluorescence energy means that replacing a fluorescent acceptor like TAMRA with a quencher such as Iowa Black TM FQ will result in an oligonucleotide construct that has no measurable fluorescence as long as the oligonucleotide tether remains intact.
  • Such constructs can greatly simplify many fluorescence assays since they do not have background fluorescence.
  • fluorophore - quencher dual - labeled probes have become a standard in kinetic (real-time) PCR. Quenchers absorb fluorophore emission energies over a wide range of wavelengths. This expanded dy namic range greatly adds to the utility of fluorescence quenchers, particularly in the case of multiplexing assays with different fluorophores [Integrated DNA Technologies, 2002].
  • Multiplexing is defined as the screening of multiple targets, the use of different reporter genes within the same experiment, or the combination of many signals into a single transmission circuit or channel [Miret et al., 2005]. Multiplexing allows for the parallel processing of multiple targets and promises to increase efficiencies, reduce costs, and improve the quality and content of screening data. In addition, it can also simplify the construction and execution of target selectivity panels.
  • the use of multiplexing in hit discovery has been previously reported for kinases, nuclear receptors, and GPCRs. Common strategies of cell-based multiplexing assay approaches are combinations of glow- and flash-light reporter genes or reporter genes with different substrate specificity.
  • Fluorogenic nuclease assays are a real time quantitation method that uses a probe to monitor formation of amplification product.
  • the basis for this method of monitoring the formation of amplification product is to measure continuously PCR product accumulation using a dual-labelled fluorogenic oligonucleotide probe, an approach frequently referred to in the literature simply as the "TaqMan method".
  • the probe used in such assays is typically a short (about 20-25 bases) oligonucleotide that is labeled with two different fluorescent dyes.
  • the 5' terminus of the probe is attached to a reporter dye and the 3' terminus is attached to a quenching dye, although the dyes could be attached at other locations on the probe as well.
  • the probe is designed to have at least substantial sequence complementarity with the probe binding site. Upstream and downstream PCR primers which bind to flanking regions of the locus are added to the reaction mixture. When the probe is intact, energy transfer between the two fluorophors occurs and the quencher quenches emission from the reporter.
  • the probe is cleaved by the 5' nuclease activity of a nucleic acid polymerase such as Taq polymerase, thereby releasing the reporter from the oligonucleotide- quencher and resulting in an increase of reporter emission intensity which can be measured by an appropriate detector.
  • Computer software is capable of recording the fluorescence intensity of reporter and quencher over the course of the amplification. The recorded values will then be used to calculate the increase in normalized reporter emission intensity on a continuous basis. The increase in emission intensity is plotted versus time, i.e., the number of amplification cycles, to produce a continuous measure of amplification.
  • the amplification plot is examined at a point during the log phase of product accumulation. This is accomplished by assigning a fluorescence threshold intensity above background and determining the point at which each amplification plot crosses the threshold (defined as the threshold cycle number or Ct or CP). Differences in threshold cycle number are used to quantify the relative amount of PCR target contained within each tube. Assuming that each reaction functions at 100% PCR efficiency, a difference of one Ct represents a two-fold difference in the amount of starting template. The fluorescence value can be used in conjunction with a standard curve to determine the amount of amplification product present.
  • These detection methods involve some alteration to the structure or conformation of a probe hybridized to the locus between the amplification primer pair.
  • the alteration is caused by the template-dependent extension catalyzed by a nucleic acid polymerase during the amplification process.
  • the alteration generates a detectable signal which is an indirect measure of the amount of amplification product formed.
  • some methods involve the degradation or digestion of the probe during the extension reaction. These methods are a consequence of the 5'-3' nuclease activity associated with some nucleic acid polymerases.
  • Polymerases having this activity cleave mononucleotides or small oligonucleotides from an oligonucleotide probe annealed to its complementary sequence located within the locus.
  • the 3' end of the upstream primer provides the initial binding site for the nucleic acid polymerase.
  • the nucleic acid polymerase displaces a portion of the 5' end of the probe and through its nuclease activity cleaves mononucleotides or oligonucleotides from the probe.
  • the upstream primer and the probe can be designed such that they anneal to the complementary strand in close proximity to one another. In fact, the 3' end of the upstream primer and the 5' end of the probe may abut one another. In this situation, extension of the upstream primer is not necessary in order for the nucleic acid polymerase to begin cleaving the probe.
  • nucleic acid polymerase In the case in which intervening nucleotides separate the upstream primer and the probe, extension of the primer is necessary before the nucleic acid polymerase encounters the 5' end of the probe. Once contact occurs and polymerization continues, the 5'-3' exonuclease activity of the nucleic acid polymerase begins cleaving mononucleotides or oligonucleotides from the 5' end of the probe. Digestion of the probe continues until the remaining portion of the probe dissociates from the complementary strand.
  • the two end sections can hybridize with each other to form a hairpin loop.
  • the reporter and quencher dye are in sufficiently close proximity that fluorescence from the reporter dye is effectively quenched by the quencher dye.
  • Hybridized probe in contrast, results in a linearized conformation in which the extent of quenching is decreased.
  • a variety of options are available for measuring the amplification products as they are formed.
  • One method utilizes labels, such as dyes, which only bind to double stranded DNA.
  • amplification product which is double stranded
  • dyes it is possible to distinguish between dye molecules free in solution and dye molecules bound to amplification product.
  • certain dyes fluoresce only when bound to amplification product. Examples of dyes which can be used in methods of this general type include, but are not limited to, Syber Green.TM. and Pico Green from Molecular Probes, Inc.
  • Molecule beacons enable dynamic, real - time detection of nucleic acid hybridization events both in vitro and in vivo.
  • One of the primary advantages of molecular beacons is that they can discriminate between targets that differ by as little as a single base pair change, making them ideal for investigating single nucleotide polymorphisms (SNPs).
  • Molecular beacons are designed so that probe sequence is sandwiched between complementary sequences that form the hairpin stem.
  • Molecular beacons must be designed so that the transition between two conformational states - the hairpin and the probe: target duplex is thermodynamically favorable. The temperature and the buffer used will influence probe specificity and must be carefully controlled.
  • the melting temperature of both the hairpin structure and the probe:target duplex shou Id be 7-10 oC higher than the temperature used for detection or for primer annealing.
  • a perfect match probe- target hybrid will be energetically more stable than the stem-loop structure whereas a mismatched probe target hybrid will be energetically less stable than the stem-loop structure.
  • This characteristic is the basis of the extraordinary specificity offered by molecular beacons. Specificity can also be relaxed by making the probe sequence in the loop and the probe - target hybrid more stable [Integrated DNA Technologies, 2002].
  • Scorpions probes consist of a primer covalent tly linked to a spacer region followed by a probe that contains a fluorophore and a quencher.
  • the probe contains a specific, complementary target sequence, a spacer region which forms a self - complementary stem, a fluorophore, and an internal quencher all contiguous with the primer.
  • the probe When not bound to the target, the probe remains in a stem - loop structure which keeps the quencher and fluorophore proximal and allows the quencher to absorb the fluorescence emitted from the fluorophore.
  • the primer will bind to the target and go through the first round of target synthesis.
  • the probe will be attached to the newly synthesized target region.
  • the spacer region prevents the DNA polymerase from copying the probe region and disrupting the stem structure.
  • next-generation sequencing offers high- throughput gene expression profiling, genome annotation or discovery of non-coding RNA. Sequence decoding is usually performed using dideoxy chain termination technology. With increasing importance of DNA sequencing in research and diagnostics, new methods were developed allowing a high- throughput sample treatment.
  • SBS sequencing-by-synthesis
  • pyrosequencing The important technological developments, summarized under the term next-generation sequencing, are based on the sequencing-by-synthesis (SBS) technology called pyrosequencing.
  • SBS sequencing-by-synthesis
  • pyrosequencing The transcriptomics variant of pyrosequencing technology is called short- read massively parallel sequencing or RNA-Seq [Stahl, 2013].
  • High-content screening also known as high-content analysis (HCA) or cellomics
  • HCS high-content analysis
  • HCA high-content analysis
  • cellomics is a method that is used in biological research and drug discovery to identify substances such as small molecules, peptides, or RNAi that alter the phenotype of a cell in a desired manner.
  • high content screening is a type of phenotypic screen conducted in cells. Phenotypic changes may include increases or decreases in the production of cellular products such as proteins and/or changes in the morphology (visual appearance) of the cell.
  • High content screening includes any method used to analyze whole cells or components of cells with simultaneous readout of several parameters.
  • cells are first incubated with the substance and after a period of time, structures and molecular components of the cells are analyzed.
  • the most common analysis involves labeling proteins with fluorescent tags, and finally changes in cell phenotype are measured using automated image analysis.
  • fluorescent tags with different absorption and emission maxima, it is possible to measure several different cell components in parallel.
  • the imaging is able to detect changes at a subcellular level (e.g., cytoplasm vs. nucleus vs. other organelles) [Alanine, 2008; Haskins, 2004].
  • the test compound is preferably a small molecule which binds to and occupies the active site of target polypeptide, thereby making the ligand binding site inaccessible to substrate such that normal biological activity is prevented.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules.
  • Potential ligands which bind to a polypeptide or cell include, but are not limited to, the natural ligands of known target and analogues or derivatives thereof.
  • either the test compound can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • a detectable label such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • Detection of a test compound which is bound to the target polypeptide or cell can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.
  • binding of a test compound to target polypeptide or cell can be determined without labeling either of the interactants.
  • BIA Bimolecular Interaction Analysis
  • SPR optical phenomenon surface plasmon resonance
  • Luciferases are commonly used as reporter genes in cell based assay formats for the characterization of second messenger and promotor activity modulators. These assays are used to test and characterize the activity of compounds or whole compound libraries. Often recombinant cell lines with luciferases under the control of second messenger responsive elements are used. Depending on the used luciferase the cell supernatant or cell lysate is used for activity testing. Usually the microtiter plate will be discarded after the measuring.
  • the cell lysate of the completed luminescence measurement could be used as a template lysate for a QPCR measurement without any further processing.
  • the lysate or an aliquot will be transferred to a QPCR useable microtiter plate. After addition of all reagent the QPCR could be performed.
  • Both readouts can be used to analyse the signaling cascade of a given target gene at different levels using the same sample, with one combinatorial assay set-up.
  • the modulation of the cAMP amount in a cell can be analysed with a reporter gene-based readout applying a cAMP responsive genetic element, whereas the activity of downstream regulated target genes may be detected by a subsequent QPCR assay within the same sample.
  • a cell based assay was performed as described in example 3.
  • the cells were incubated with increasing amounts of forskoline and incubated for the given time.
  • the cell lysate was used for the qualification of firefly luciferase mRNA.
  • Fig. 2 the relative light units for the different concentrations of forskoline are shown.
  • the luminescence was increased with increased concentrations of forskoline.
  • a part of the cell lysate was transferred to a QPCR microtiter plate.
  • the QPCR was performed as described in example 3.
  • the relative expression was calculated as described in example 2 and is shown in Fig. 1.
  • the relative expression of the firefly luciferase was increased with increasing concentrations of forskoline.
  • a cell based assay was performed as described in example 6. The cells were incubated with increasing amounts of JQl and incubated for the given time. After the luminescence measurement the cell lysate was used for the qualification of nanoluc luciferase mRNA. In Fig. 8 the relative light units for the different concentrations of JQl are shown. The luminescence was increased with increased concentrations of JQl .
  • RNA quantification or analysis of gene expression were combined with reporter gene based methods. Those methods are described, but not limited to, under “reporter genes", “expression analysis” and “multiplexing reporter gene assays”. The combination of methods is independent from the used microtiter plate format and could be used, but not limited to, 96, 384, 1536 and 6144 well formats.
  • QPCR is based on a sequence of biochemical based reactions, the efficiency and accuracy of which are greatly affected by the applied experimental conditions. All steps of the RNA quantification could be effected, for example (but not limited to): reverse transcription, DNA annealing, PCR and fluorescence detection.
  • Magnesium from the reporter gene assays has an influence on the transcriptional analysis assay (in particular QPCR).
  • Magnesium is a cofactor of firefly luciferase and necessary for a high sensitivity firefly luciferase based reporter gene assay [Zako, 2003].
  • the activity of the firefly luciferase directly depends on the Magnesium concentration in the reporter gene assay buffer (shown in Fig. 4 of [Zako, 2003]).
  • Magnesium is an important cofactor for PCR reactions which directly influences the accuracy with which the PCR primers hybridize with their template.
  • the concentration of Magnesium in PCR or QPCR reaction buffers therefore is a key parameter determining the results of the methods. Consequently, the optimization of PCR or QPCR processes mainly relates to the determination of the optimal amounts of Magnesium within the reaction solutions [Roux, 2009].
  • Luciferase is an intracellularly located enzyme. To measure the luminescence as a proportion of firefly luciferase activity it is therefore necessary to disrupt the cells in order to allow an interaction between the enzyme and its extracellularly located substrate. Cells are usually disrupted by the use of detergents. The concentration and type of detergent is cell and assay dependent [Brown, 2008; Schuck, 2003]. However, it is known in the art that the presence of detergents and the concentration of salts has a direct effect on QPCR efficacy [Shatzkes, 2014; Fig. 1].
  • Example 1 Design of primer probes for L32, Firefly luciferase, Nanoluc luciferase
  • cell lysates were transferred to 384-well or 1536-well QPCR microtiter plates. For 384-well 2 ⁇ L and for 1536-well 1 ⁇ cell lysate were transferred. The preparation of the cell lysates are described in examples 3-6.
  • the Custom qPCR kit from Eurogentec was used according to the manufacturer's specifications by using the following specifications.
  • the qPCR was performed in at total volume of 10 ⁇ per well with 2 ⁇ lysate, 1.5 ⁇ primer/probe mix (1.33 ⁇ each), 2.5 ⁇ master mix, 0.05 ⁇ enzyme mix and 1.45 ⁇ water
  • RNA Virus Master kit from Roche was used according to the manufacturer's specifications by using the following specifications.
  • the qPCR was performed in at total volume of 2 ⁇ per well with ⁇ ⁇ . lysate, 300 nL primer/probe mix (1.33 ⁇ each), 400 nL master mix, 40 nL enzyme mix and 260 nl water
  • CP value (as given from LC480 or LC1536 respectively) of Nanoluc luciferase: CP-Nano
  • CHO cells per well were seeded on 384-well microtiter plates in 50 ⁇ . complete growth medium. The cells are incubated for 24 hours under standard cell culture conditions (37 °C, 5% C02). After 24h the supernatant is removed and the compound is added Buffer X in the given final concentrations. The cell are incubated for 2 hours under standard conditions. The supernant is removed and ⁇ ⁇ buffer A (+ 10 U/well RNase Inhibitor) + RNase free water are added. The luminescence measuring is immediately started using a standard luminometer. The luminescence signal is measured for 60sec and integrated for 1 sec . The relative light units are shown in Fig. 2.
  • the cell lysate could directly be used for RNA or DNA quantification or analysis.
  • 2 ⁇ of the lysate is transferred to a 384well QPCR microtiter plate and the prepared Master Mix is added.
  • the cDNA synthesis is performed for 30 min at 48°C .
  • the QPCR is performed using a LC480 (Roche) with the following protocol: 10 min at 95°C, followed by 50 cycles of melting at 95°C for 15 sec and annealing/extending at 60°C for 1 min.
  • the relative expression is calculated as described under example 2.
  • the relative expression of the firefly luciferase is shown in Fig. 1.
  • Buffer A 50mM Tris(hydroxymethyl)-aminometha (pH 8), 40mM Natrium chloride, 1.5 mM Magnesium chloride, 0.5% Octylphenoxy poly(ethyleneoxy)ethanol (IGEPAL® CA-630), 50 mM Guanidiniumthiocyanat, 23.85 mM Adenosintriphosphat, 1.215 mM Acetyl-CoA, 2.115 mM D- Luciferin
  • Buffer X 2mM Calcium chloride, 20mM 2-[4-(2-hydroxyethyl)piperazin-l -yl]ethanesulfonic acid, 130mM Natrium chloride, 5mM Kalium chloride, 5mM Sodium hydrogen carbonate, 2mM Magnesium chloride hexahydrate
  • Example 4 Combination of firefly luciferase with QPCR - protocol 2 in 384well format
  • CHO cells per well were seeded on 384well microtiter plates in 50 ⁇ . complete growth medium .
  • the cells are incubated for 24 hours under standard cell culture conditions (37 °C, 5% C02). After 24h the supernatant is removed and the compound is added Buffer X in the given final concentrations.
  • the cell are incubated for 2 hours under standard conditions.
  • the supernant is removed and ⁇ ⁇ buffer B (+ 10 U/well RNase Inhibitor) + RNase free water are added.
  • the luminescence measuring is immediately started using a standard luminometer.
  • the luminescence signal is measured for 60sec and integrated for 1 sec .
  • the relative light units are shown in Fig. 2.
  • the cell lysate could directly be used for RNA or DNA quantification or analysis.
  • 2 ⁇ of the lysate is transferred to a 384well QPCR microtiter plate and the prepared Master Mix is added.
  • the cDNA synthesis is performed for 30 min at 48°C .
  • the QPCR is performed using a LC480 (Roche) with the following protocol: 10 min at 95°C, followed by 50 cycles of melting at 95°C for 15 sec and annealing/extending at 60°C for 1 min. .
  • the relative expression is calculated as described under example 2.
  • the relative expression of the firefly lucif erase is shown in Fig. 3. Used buffers:
  • Buffer B 120mM N-(Tri(hydroxymethyl)methyl)glycin, 15.6 mM Magnesium chloride, 1.56 mM Ethylendiamintetraacetat, 198 mM Dithiothreitol, 31.8 mM Adenosintriphosphat, 1.62 mM Acetyl- CoA, 2.82 mM Luciferin
  • CHO cells per well were seeded on 384well microtiter plates in 50 ⁇ . complete growth medium. The cells are incubated for 24 hours under standard cell culture conditions (37 °C, 5% C02). After 24h the supernatant is removed and the compound is added Buffer X in the given final concentrations. The cell are incubated for 2 hours under standard conditions. The supernant is removed and ⁇ ⁇ buffer C (+ 10 U/well RNase Inhibitor) + RNase free water are added. The luminescence measuring is immediately started using a standard luminometer. The luminescence signal is measured for 60sec and integrated for 1 sec . The relative light units are shown in Fig. 2.
  • the cell lysate could directly be used for RNA or DNA quantification or analysis.
  • 2 ⁇ of the lysate is transferred to a 384well QPCR microtiter plate and the prepared Master Mix is added.
  • the cDNA synthesis is performed for 30 min at 48°C .
  • the QPCR is performed using a LC480 (Roche) with the following protocol: 10 min at 95°C, followed by 50 cycles of melting at 95°C for 15 sec and annealing/extending at 60°C for 1 min. .
  • the relative expression is calculated as described under example 2.
  • the relative expression of the firefly luciferase is shown in Fig. 5. Used buffers:
  • Buffer C Luciferase Assay System (El 500, Promega) Assay protocol:
  • CHO cells per well were seeded on 1536-well microtiter plates in 1 L complete growth medium .
  • the cells are incubated for 24 hours under standard cell culture conditions (37 °C, 5% C02). After 24 h Buffer X is added with compounds in the given concentrations.
  • the cell are incubated for 6 hours under standard conditions.
  • the supernant is removed and 1 ⁇ ⁇ buffer D is added.
  • the luminescence measuring is immediately started using a standard luminometer.
  • the luminescence signal is measured for 60 sec and integrated for 1 sec .
  • the relative light units are shown in Fig. 2.
  • buffer E (containing 1 U/well RNase Inhibitor) is added.
  • the cell lysate could directly be used for RNA or DNA quantification or analysis.
  • 1 ⁇ of the lysate is transferred to a 1536-well QPCR microtiter plate and the prepared Master Mix is added.
  • the cDNA synthesis is performed for 30 min at 48°C .
  • the QPCR is performed using a LC1536 (Roche) with the following protocol: 10 min at 95°C, followed by 50 cycles of melting at 95°C for 15 sec and annealing/extending at 60°C for 1 min. .
  • the relative expression is calculated as described under example 2.
  • the relative expression of the nanoluc luciferase is shown in Fig. 7. Used Buffers:
  • Buffer D Nano-Glo® Luciferase Assay (Nl 130, Promega)
  • Buffer E 1 ⁇ Triton Buffer (PAA, T21-160) + 5 ⁇ RNase free water
  • Fig. 1 combination of firefly luciferase with QPCR - protocol 1 (expression)
  • Fig. 2 combination of firefly luciferase with QPCR - protocol 1 (luminescence)
  • Fig. 3 combination of firefly luciferase with QPCR - protocol 2 (expression)
  • Fig. 4 combination of firefly luciferase with QPCR - protocol 2 (luminescence)
  • Fig. 5 combination of firefly luciferase with QPCR - protocol 3 (expression)
  • Fig. 6 combination of firefly luciferase with QPCR - protocol 3 (luminescence)
  • Fig. 7 combination of nanoluc luciferase with QPCR - protocol 4 (expression)
  • Fig. 8 combination of nanoluc luciferase with QPCR - protocol 4 (luminescence)
  • Fig. 9 spectra of selected fluorescent labels
  • Fig. 10 absorption and fluorescnce spectrum of firefly luciferase substrate
  • Fig. 1 shows the relative expression of firefly luciferase using protocol 1 as described under example 3.
  • ⁇ relative expression
  • Fig. 2 shows the relative light units (RLU) of firefly luciferase using protocol 1 as described under example 3.
  • ⁇ Total Light emitted by the luciferase-catalyzed chemiluminescent reaction, in relative light units (RLU), measured 60 seconds with integration period of 1 second
  • Fig. 3 shows the relative expression of firefly luciferase using protocol 2 as described under example 4.
  • Fig. 4 shows the relative light units (RLU) of firefly luciferase using protocol 1 as described under example 4.
  • ⁇ Total Light emitted by the luciferase-catalyzed chemiluminescent reaction, in relative light units (RLU), measured 60 seconds with integration period of 1 second
  • Fig. 5 shows the relative expression of firefly luciferase using protocol 1 as described under example 5.
  • Fig. 6 shows the relative light units (RLU) of firefly luciferase using protocol 1 as described under example 5.
  • B Total Light emitted by the luciferase-catalyzed chemiluminescent reaction, in relative light units (RLU), measured 60 seconds with integration period of 1 second Fig.
  • Fig. 8 shows the relative light units (RLU) of nanoluc luciferase using protocol 4 as described under example 6.
  • ⁇ Total Light emitted by the luciferase-catalyzed chemiluminescent reaction, in relative light units (RLU), measured 60 seconds with integration period of 1 second
  • Fig. 9 shows the fluorescence spectra of selected fluophores which could be used for probe labeling in QPCR. (from Fig. 1 [Van Poucke, 2012]).
  • Fig. 10 shows the absorption and fluorescnce spectrum of firefly luciferase substrate (from Fig. 1 [Hiyama, 2012]) Listing of Sequences
  • SEQ ID NO:4 forward primer firefly luciferase
  • SEQ ID NO: 6 probe firefly luciferase
  • SEQ ID NO: 7 forward primer nanoluc luciferase
  • SEQ ID NO: 8 reverse primer nanoluc luciferase
  • SEQ ID NO: 9 probe nanoluc luciferase
  • SEQ ID NO: l 1 nucleotide sequence of firefly luciferase
  • SEQ ID NO: 12 nucleotide sequence of nanoluc luciferase

Abstract

The present invention relates to a method for analysing the effects of one or more test compounds on cells or tissues, comprising at least a step comprising a cell-based assay, and a step comprising an expression analysis related to said cells or tissues.

Description

COMBINATION OF REPORTER GENE ASSAYS AND TRANSCRIPTIONAL ANALYSIS
The present invention relates to the field of molecular and cell biology, more particularly, the present invention relates to a method which combines two or more techniques to analyse the effects of compounds on cells or tissues. The invention also relates to a method which combines methods for the analysis or quantification of RNA with methods to measure second messenger signaling or promotor activity.
Background of the invention
The modern drug research process has reversed the classical pharmacological strategy. Today, research programs are initiated based on biological evidence suggesting a particular gene or gene product to be a potential target for small molecule drugs useful for therapy. Also, it allows setting up a linear drug discovery process starting from target identification to finally delivering molecules for clinical development. One central element is lead discovery through High-Throughput- Screening (HTS) of comprehensive corporate compound libraries. Pharma research in most organizations is organized in discrete phases together building a "value chain" along which discovery programs proceed and which ultimately result in the identification of a drug candidate that enters clinical testing. Following a technical assesment of the targets' "drugability" [Groom, 2002], the probability to identify small molecule modulators, and the technical feasibility to perform an HTS, target-specific assays are developed to probe the corporate compound collection for promising lead candidates. Lead discovery in the pharmaceutical industry today still depends largely on experimental screening of compound libraries. Accordingly, the industry has invested heavily in expanding these libraries and established appropiate screening capabilities to handle large numbers of compounds within a reasonable period of time. HTS involves laboratory automation to handle the different assay steps typically performed on microtiter plates and has witnessed remarkable developments over time. Assay technologies have advanced to provide a large variety of various cell-based and biochemical test formats for a large spectrum of disease-relevant target classes. In parallel, further miniaturization of assay volumes and parallelization of processes have further increased the assay throughput. HTS is performed, for example, in 1536-well plates with assay volumes between 5-10 μΐ. This set up, together with fully-automated robotic systems allows for testing in excess of 200,000 compounds per day. Comprehensive substance collections together with sophisticated screening technologies have resulted in a clear advantage in lead discovery especially for poorly draggable targets. The productivity of HTS has recently been questioned, because often no modulators could be found for a given target, and the poor drugability of many molecular targets pursued in research programs, together with a poor quality of compounds in screening libraries, have been identified as major drawbacks responsible for low success. Improvements in the design of library screening methods aim at the identification of compounds with more "drug-like" physico-chemical properties, like oral availability. Summary of the invention
These and further objects are met with methods and means according to the independent claims of the present invention. The dependent claims are related to specific embodiments.
Embodiments of the invention
Before the invention is described in detail, it is to be understood that this invention is not limited to the particular component parts or structural features of the devices or compositions described or process steps of the methods described as such devices and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include singular and/or plural referents unless the context clearly dictates otherwise. Further, in the claims, the word "comprising" does not exclude other elements or steps. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.
It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.
It is further to be understood that embodiments disclosed herein are not meant to be understood as individual embodiments which would not relate to one another. Features discussed with one embodiment are meant to be disclosed also in connection with other embodiments shown herein. If, in one case, a specific feature is not disclosed with one embodiment, but with another, the skilled person would understand that does not necessarily mean that said feature is not meant to be disclosed with said other embodiment. The skilled person would understand that it is the gist of this application to disclose said feature also for the other embodiment, but that just for purposes of clarity and to keep the specification in a manageable volume this has not been done. Furthermore, the content of the prior art documents referred to herein is incorporated by reference. This refers, particularly, to prior art documents that disclose standard or routine methods. In that case, the incorporation by reference has mainly the purpose to provide sufficient enabling disclosure, and avoid lengthy repetitions.
According to one embodiment of the invention, a method for analysing the effects of one or more test compounds on cells or tissues is provided, comprising at least
(i) a step comprising a cell-based assay, and
(ϋ) a step comprising an expression analysis related to said cells or tissues.
As will be shown herein, the combination of a cell-based assay and an expression analysis readout has a number of unexpected advantages, namely, saving of time, saving of resources, enhanced assay accuracy, and combination of data from different pathway levels.
As used herein, the term "cell-based assay" relates to methods using defined cells, which are then exposed the test compounds.
Assays developed for compound screening can be divided very broadly into two categories, namely (i) biochemical assays and (ii) cell-based assays. Biochemical assays are target-based in vitro assays which historically have been the mainstay of high throughput screening (HTS) in the pharmaceutical industry. Such assays include assessment of enzymatic activity (e.g. for kinases, proteases, or transferases), receptor-ligand binding (e.g., for G-protein coupled receptors (GPCRs), ion channels , or nuclear receptors), or protein-protein interactions. Biochemical assays are often direct and specific to the target of interest and can be miniaturized readily. However, not all targets can be purified or prepared in a manner suitable for biochemical measurement. Additionally, a certain activity of a small molecule observed in an in vitro assay does not always translate into the same activity in a cellular context, because of issues including membrane permeability, off-target effects, and cytotoxicity [Tolliday, 2009].
In contrast thereto, cell-based assays do not require an a priori knowledge of the target that might be affected by the test compounds. Further, in contrast to biochemical assays, the likelihood that a extrapolation from assay results into a living system can be done is much higher. In most cell based assays, entire pathways of interest can be interrogated, providing the opportunity for multiple potential intervention points, as opposed to a single predefined step with the biochemical approach. Examples for cell based assays formats are (but not limited to) proliferation assays, reporter gene assays, second messenger assays and high content screening assays. In such way, parameters such as potential drug cytotoxicity, mechanism of action, and biological activity can be determined. These types of analysis provide a cost-effective and reliable indication of drug activity in a living system and can be used to measure the chosen aspects over time. As used herein, the term "expression analysis" relates to a method involving the qualitative or quantitative determination of the expression of one gene, or a set of gene, either measured on the protein level or on the mRNA level.
Expression analysis can be done with any of the following methods:
• microarrays
• quantitative polymerase chain reaction (QPCR, RT-PCR)
• SAGE (Serial Analysis of Gene Expression)
• EST/cDNA library
· Northern Blots
• Nucleic acid sequencing
Acccording to one embodiment of said method according to the present invention, the cell-based assay format is based on measuring second messenger signaling or promotor activity [Roda, 2010].
Acccording to another embodiment of said method according to the present invention, said cell- based assay format is a reporter gene based assay format.
In cell-based assays, cells can be engineered to express specific gene products in response to a given stimulus. The gene product itself may possess an inherent property that enables it to be measured directly, e.g., green fluorescent protein (GFP), or it may display enzymatic activity that can be monitored, e.g., luciferase. Alternatively, the gene product may respond to changes in the levels of a signaling molecule, e.g., the Ca 2+ ion-mediated activation of aequorin luminescence. The continuing development of genetically engineered vectors, coupled with powerful detection techniques, allows many cell types to be engineered as biodetectors for myriad classes of biochemical and signaling pathways.
When establishing an assay system, a number of factors need to be considered in order to optimize the assay. The choice of a particular promoter, the number of promoter copies per reporter gene unit, and the nature of the reporter gene allow to control the basal level of reporter gene activityand control of the degree of stimulation measured. Endogenous promoters, such as c-fos, the cAMP response element (CRE), or the estrogen response element are commonly used. However, the accuracy of assays employing these promoters istheir activation through endogenous intracellular signaling events. The reporter gene itself should ultimately generate a signal that can be clearly identified. The reporter gene products can be either intracellular or extracellular in nature. Intracellular products are retained in the cell for quantification in situ or following cell lysis.
Extracellular products are secreted into the extracellular medium for assay, allowing repeated experimentation and sampling without disrupting the cells. Commonly used intracellular reporter genes are chloramphenicol acetyltransferase (CAT), β -galactosidase, luciferase, aequorin, and GFP. Extracellular reporter genes are usually secreted placental alkaline phosphatase (SPAP) or β - lactamase [Tolliday, 2009]. Preferably, a reporter gene is chosen the gene product of which can easily be detected with standard biochemical or histochemical methods. Two commonly used types of reporter genes are resistence genes and reporter genes.
1. Resistance genes are genes, the expression of which confers on a cell the resistance to antibiotics or other substances which would, in the absence of the resistence gene, cause the death of the cell.
2. Using genetic engineering technologies cell lines are modified and express reporter proteins under the control of recombinant or endogenous promotors. Those reporter genes or proteins can be fused to a target genes to detect expression thereof by luminescent or fluorescent readouts (e.g. GFP, luciferase), namely because the reporter gene is coexpressed with the target gene.
Acccording to another embodiment of said method according to the present invention, said cell- based assay format is at least one selected from the group consisting of:
• a luminescence based assay format,
• a fluorescent dye based assay format
• a FRET (Fluorescence Resonance Energy Transfer) based assay format, and/or
• a Fluorescence polarisation / anisotropy based assay format Luminescence is the term given to the emission of photons in the visible spectral range, with this emission being brought about by excitated emitter molecules. In contrast to fluorescence, the energy for this is not supplied externally in the form of radiation of shorter wavelength.
A distinction is made between chemiluminescence and bioluminescence. Chemoluminescence is the term given to a chemical reaction which leads to an excited molecule which itself emits light when the excited electrons return to the normal energy level. Bioluminescence is the term used when this reaction is catalyzed by an enzyme. The enzymes which participate in the reaction are generally termed luciferases. Luciferases are peroxidases or monooxygenases and dioxygenases. The enzyme substrates, which form the starting substances for the light- emitting products, are termed luciferins. They differ from species to species. The quantum yield of the systems lies between 0.1 and 0.9 photons per transformed substrate molecule. Luciferases can be classified on the basis of their origin or their enzymic properties. Luciferases can also be distinguished from each other on the basis of their substrate specificity. The most important substrates include coelenterazine and luciferin, and also derivatives of the two substances. An overview, but not limited to, of some luciferases:
• Lux Genes (Vibrio fischerii) - substrate: FMN, Dodecanal, NADH
• Renilla Luciferase (Renilla reniformis) - substrate: Coelenterazine
• Vargula / Cypridia Luciferase (Vargula hilgendorferii) - substrate: Vargula Luciferin
• Watasemia Luciferase (Watasenia scintillans) - substrate: Watasemia Luciferin
• Olophorus Luciferase (Olophorus gracilirostris) - substrate: Coelenterazine
• Aequorin (Aequoria aequoria) - substrate: Coelenterazine
• Obelin (Obelia) - substrate: Coelenterazine
• Firefly Luciferase (Photinus pyralis) - substrate: Firefly Luciferin
• Nanoluc [Engineered9 (Oplophorus gracilirostris) - substrate: Furimazine
• Clytin and mtClytin (Cyltia gregarai) - substrate: Coelenterazine
As regards fluoresence based assays, quite a few proteins are known and available to day which emit light when excited with light from a given spectral range. Aequoria victoria aequorin, which was identified as the first light-producing protein in 1962, emits, as an isolated protein, a blue light. Later, the green fluorescent protein (GFP) was isolated from Aequoria victoria, which, owing to the excitation by aequorin, makes the medusa appear green phenotypically. Green fluorescent proteins have been isolated from different organisms. These include Hydozoa (aequoria, halistaura obelia) and anthropods (acanthotilum, sea cactus, cavernularia, renila, ptilosarcus, stylatula). An overview, but not limited to, of some fluorescent proteins, is given in the following: · Green fluorescent protein (Aequorea macrodactyla, Gene ID: AF435433)
• Green fluorescent protein (Aequoria, Gene ID: L29345)
• Green fluorescent protein- like protein (Agaricia agaricites, Gene ID: AY037775)
• Green fluorescent protein- like protein (Agaricia fragilis, Gene ID: AY037765)
• Green fluorescent protein (Dendronephthya, Gene ID: AF420591)
· Red fluorescent protein (Entacmaea quadricolor, Gene ID: AY130757)
• Green fluorescent protein- like protein (Caribbean anemone, Gene ID: AY037777)
• Green fluorescent protein (Heteractis crispa, Gene ID: AF420592)
• Green fluorescent protein- like protein (Montastraea annularis, Gene ID: AY037766)
• Green fluorescent protein-like protein (Montastraea cavernosa, Gene ID: AY037768)
· Cyan fluorescent protein (Montastraea cavernosa, Gene ID: AY056460)
• Green fluorescent protein (Renilla muelleri, Gene ID: AY015996)
• Green fluorescent protein (Renilla renoformis, Gene ID: AF372525)
• Green fluorescent protein- like protein (Ricordea florida, Gene ID: AY037774) The fluorescent proteins differ from one another not only due to their nucleotide and amino acid sequences but also due to their biochemical and physical properties. The spectral characteristics of the fluorescent proteins may differ both on the side of excitation and on the side of emission.
It was shown that it is possible to alter the physical and biochemical properties of fluorescent proteins by altering the amino acid sequence thereof. Examples of mutagenized fluorescent proteins have been described in the literature [Youvan, 1995; Tsien, 1996]. Fluorescent proteins are already used in a wide variety of areas. The use of fluorescent proteins in "Fluorescence Resonance Energy Transfer" (FRET), "Bio luminescence Resonance Energy Transfer (BRET) and other energy transfer methods has already been described in the literature [Youvan, 1996] . Fluorescence-based assays can be generally divided into two classes. The first class encompasses techniques that macroscopically detect the total fluorescence intensity, fluorescence polarization, fluorescence resonance energy transfer (FRET), fluorescence lifetime, time-resolved fluorescence, and combinations of these techniques, such as time-resolved fluorescence polarization. The second class of fluorescence-based assays detects fluorescence from single fluorescent molecules, such as fluorescence correlation spectroscopy and fluorescence intensity distribution analysis. These fluorescence techniques have been used to monitor an enormous collection of biological processes, such as macromolecule-macromolecule interactions, macromolecule-small molecule interactions, enzymatic activities, signal transduction, cell health, and states and locations of molecules, organelles, or cells [Tolliday, 2009] .
Fluorescence resonance energy transfer (FRET) utilises non-radiative energy transfer from a donor fluorophore to an acceptor fluorophore resulting in fluorescence emission from the acceptor. FRET can occur where there is sufficient overlap of the donor emission spectrum with the acceptor excitation spectrum, close proximity (<100 A) and correct donor-acceptor orientation of dipole moments. These parameters are readily adaptable to ligand binding assays, where binding of a fluorescent ligand with suitable spectral properties to a fluorophore-tagged protein provides the close proximity required for FRET. FRET assays require the target protein to be tagged with a donor or acceptor fluorophore, this can be achieved by fusing the N-terminus of the protein with a fluorescent protein (such as EGFP) [Pfleger, 2015] .
To monitor the binding of a fluorescent ligands to proteins on the cell surface fluorescence anisotropy (FA) could be used [Turcatti, 1995]. FA, which can also be referred to as fluorescence polarisation (FP) depending on the equation used, is based on measuring the ability of a fluorescent molecule to maintain the polarisation of light. If not bound to a protein and free in solution, the free rotation of the fluorescent ligand means that the direction of the emitted light will not be at the same angle as the polarised light used to excite the fluorophore. Whereas, if the ligand is bound to a protein and is therefore in a fixed position, the light will be emitted in the same plane as the original excitation and maintain the polarisation. This difference in polarisation can be used to differentiate bound from free ligand. As FA and FP can distinguish between bound and unbound ligand, there is no requirement for a wash or filtration step. It is therefore a homogenous assay and is normally performed on cell membranes. Due to its homogenous nature, experiments can theoretically be undertaken using FA or FP to measure the kinetic parameters of ligand binding. However, it should be pointed out that a high degree of ligand depletion is normally a consequence of the high receptor concentrations and low ligand concentrations required to get a significant change in FA/FP on ligand binding [Pfleger, 2015]. Acccording to another embodiment of said method according to the present invention, the expression analysis of said cells or tissues is a transcriptome analysis.
The transcriptome is defined as the full complement of RNA transcripts of the genes of a cell or organism. The types and relative abundance of different transcripts, i.e. the messenger RNAs (mRNAs), can be obtained by analysing cell contents using different methods. This analysis is defined as transcriptomics. Used methods for transcriptome analysis are, but not limited to, sequencing, microarrays and PCR-based methods (e.g. QPCR). Acccording to one embodiment of said method according to the present invention, the expression analysis of said cells or tissues comprises an RNA quantification. Preferably, the expression analysis of said cells or tissues comprises a QPCR or RT-PCR.
The term RT-PCR means "real-time polymerase chain reaction" while the term QPCR means "quantitative polymerase chain reaction" RT-PCR is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, i.e. in real-time, and not at its end, as in conventional PCR. Real-time PCR can be used quantitatively (Quantitative real-time PCR), semi-quantitatively (Semi quantitative real-time PCR) or qualitatively (Qualitative real-time PCR).
Two common methods for the detection of PCR products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary sequence.
Real-time PCR is carried out in a thermal cycler with the capacity to illuminate each sample with a beam of light of at least one specified wavelength and detect the fluorescence emitted by the excited fluorophore. The thermal cycler is also able to rapidly heat and chill samples, thereby taking advantage of the physicochemical properties of the nucleic acids and DNA polymerase.
The PCR process generally consists of a series of temperature changes that are repeated 25 - 50 times. These cycles normally consist of three stages: the first, at around 95 °C, allows the separation of the nucleic acid's double chain; the second, at a temperature of around 50-60 °C, allows the binding of the primers with the DNA template; [Rhoads, 1990] the third, at between 68 - 72 °C, facilitates the polymerization carried out by the DNA polymerase. Due to the small size of the fragments the last step is usually omitted in this type of PCR as the enzyme is able to increase their number during the change between the alignment stage and the denaturing stage. In addition, in four steps PCR the fluorescence is measured during short temperature phase lasting only a few seconds in each cycle, with a temperature of, for example, 80 °C, in order to reduce the signal caused by the presence of primer dimers when a non-specific dye is used. The temperatures and the timings used for each cycle depend on a wide variety of parameters, such as: the enzyme used to synthesize the DNA, the concentration of divalent ions and deoxyribonucleotides (dNTPs) in the reaction and the bonding temperature of the primers.
Original use of intercalating dyes, such as SYBR green, to monitor DNA amplification, enabled measurement of a single mRNA in a single tube. Thus, to reliably quantify expression of a single gene, it was necessary to set up multiple reactions to develop standard curves for the reference gene and the gene of interest. Another major issue with SYBR green detection is that all amplified products intercalate the dye and increase the generated signal. As a result, sensitivity of the technique made any pipetting errors as well as nonspecific amplification detrimental to the accurate estimation of expression levels. Advances in fluorescent chemistries and the use of sequence- specific probes made it possible to determine the expression of a reference gene and the gene(s) of interest in a single tube, thus requiring less sample handling and providing internal normalization. Probes used in multiplex assays are conjugated with fluorescent reporter dyes, which vary in their absorption and emission spectra. The number of genes that can be analyzed in the same tube is limited by spectral properties of the available fluorophores, which should have nonoverlapping emission spectra for separate detection. The majority of modern spectrofluorometric thermal cyclers allow the measurement of fluorescence in four different wavelength channels (although six- channel machines do exist), thereby enabling the concurrent detection of up to four different DNA targets. A choice of dyes optimized for detection in each channel is available for distinctive labelling of specific probes. TaqMan is a technique in which the release of a fluorescent reporter dye from a hybridisation probe in real-time during a polymerase chain reaction (PCR) is proportional to the accumulation of the PCR product. Quantification is based on the early, linear part of the reaction, and by determining the threshold cycle (CT), at which fluorescence above background is first detected. The most widely used probes to monitor DNA amplification in real-time PCR in clinical settings are TaqMan or hydrolysis probes [Williams, 1996]. These probes are labeled at one end with a reporter fluorescent dye and on the other with a fluorescence quencher, which must exhibit spectral overlap with the fluorophore. Quencher absorbs energy emitted by the flourophore through fluorescent resonance energy transfer (FRET); the quenchers, currently used in multiplex PCR, reemit energy as heat (dark quencher). When the probe is intact, the proximity of the quencher to the reporter dye inhibits the fluorescence signal. The Tm of the probe is usually 8-10°C higher than that of the primers, which allows the probe to anneal prior to extension. During DNA synthesis, the 5 -exonuclease activity of the Taq polymerase hydrolyses the bound probe and releases the dye from the quencher, relieving quenching effect. The level of detected fluorescence is therefore proportional to the amounts of newly synthesized DNA. A number of other types of probes are being used in real-time PCR as well, including hybridization probes and molecular beacons [Kramer, 1996]. Hybridization probes consist of two single-dye labeled oligonucleotides which bind to adjacent targets in the amplified region, thereby bringing the two dyes in close proximity and inducing FRET. Molecular beacon probes are designed as hairpins, with target sequence located in the loop and fluorescent dye and the quencher conjugated to the 5'-end and 3'-end of the oligonucleotide. In the intact, hairpin state, fluorescence is quenched; binding of the probe to the amplified sequence forces fluorophore and quencher apart, and releases fluorescent signal. Another widely used probes are Scorpion primers, which combine PCR primer and specific probe in one sequence [Little, 1999]. The structure of Scorpion primers promotes unimolecular probing mechanism; consequently, they can perform better than TaqMan probes or molecular beacons particularly under fast cycling conditions [Brown, 2000]. Described probes can be used not only to quantify levels of DNA, but to discriminate between alleles and detect mutations as well [Skoblov, 2011].
According to one embodiment of said method according to the present invention, the cell-based assay format is a luciferase based assay format, and wherein said expression analysis of said cells or tissues is a QPCR or RT-PCR. Preferably, said cells are prokaryotic or eukaryotic cells.
More preferably, said cells are selected from the group comprising human cells, animal cells, plant cells, microbial cells, and bacterial cells. According to one embodiment of said method according to the present invention, at least one of the steps is performed in at least one microtiter plate having a number of sample wells selected from the group consisting of 6, 24, 96, 384, 1536, 3072, 3456, 6144 and 9600 wells.
A microtiter plate or microplate or microwell plate or multiwell, is a flat plate with multiple "wells" used as small test tubes. The microplate has become a standard tool in analytical research and clinical diagnostic testing laboratories. A microplate typically has, but not limited to, 6, 24, 96, 384, 1536, 3072, 6144 sample wells or even more arranged in a 2:3 rectangular matrix. Some microplates have even been manufactured with 3456 or even 9600 wells, and an "array tape" product has been developed that provides a continuous strip of microplates embossed on a flexible plastic tape. Each well of a microplate typically holds somewhere between tens of nanolitres to several millilitres of liquid. Wells can be either circular or square. A number of companies have developed robots to specifically handle microplates. These robots may be liquid handlers which aspirate or dispense liquid samples from and to these plates, or "plate movers" which transport them between instruments, plate stackers which store microplates during these processes, plate hotels for longer term storage, plate washers for processing plates, plate thermal sealers for applying heat seals, de-sealers for removing heat seals, or microplate incubators to ensure constant temperature during testing. Instrument companies have designed plate readers which can detect specific biological, chemical or physical events in samples stored in these plates. Microtiter plates could be used for different cell-based assays formats, as well as expression analysis (e.g. QPCR).
Generally, the use of microtiter plates allows the screening of libraries and or the integration into High Throughput Screening (HTS) enivironments, as discussed elsewhere herein.
According to one embodiment of said method according to the present invention, a cell lysate or cell culture supernatant derived from the cell based assay step is subjected to the expression analysis step.
According to another aspect of the invention, a method of screening a library of test compounds is provided, which method encompasses a method according to the above description.
As used herein, the term "library" relates to a plurality of compounds of similar kind, e.g., a library of antibodies, a library of small molecules, or a library of aptamers. These libraries can have a size of anything between 5 to 1015 individual compounds.
According to another aspect of the invention, a kit of parts is provided which comprising reagents suitable for performing the method according to the above description.
According to another aspect of the invention, the use of such kit of parts is provided for identifying a particular gene or gene product to be a potential target for the action of potentially therapeutic agents. According to another aspect of the invention, the use of such kit of parts is provided for screening one or more test compounds which are potentially therapeutic agents, or a library thereof, for the development of a pharmaceutical drug. In said embodiment, the potentially therapeutic agents are preferably (i) biologies or (ii) small molecules. As used herein, the term "biologic" refers to (i) protein-based moelcules, (ii) nucleic-acid an based molecules or (iii) cells or cell comprising entities, all of which have a physiological effect, preferably a therapeutic effect. Molecules encompassed by that term are vaccines, cytokines, hormones, coagulation factors, antibodies and antibody-derived molecules, antibody mimetics, aptamers, RNA molecules and the like, and blood products and the like. Cells or cell comprising entities encompassed by that term are stem cells and T cells, including transgenic or chimerized variants thereof, and cellular blood products and the like.
As used herein, the term "small molecule" refers to a non-peptidic, non-oligomeric organic compound either synthesized in the laboratory or found in nature. Small molecules, as used herein, can refer to compounds that are "natural product- like", however, the term "small molecule" is not limited to "natural product-like" compounds. Rather, a small molecule is typically characterized in that it contains several carbon-carbon bonds, and has a molecular weight of less than 2500, although this characterization is not intended to be limiting for the purposes of the present invention.
Definition of further terms
An "oligonucleotide" as used herein is a stretch of nucleotide residues which has a sufficient number of bases to be used as an oligomer, amplimer or probe in a polymerase chain reaction (PCR). Oligonucleotides can be prepared from genomic or cDNA sequence and are used to amplify, reveal, or confirm the presence of a similar DNA or RNA in a particular cell or tissue. Oligonucleotides or oligomers comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 35 nucleotides, preferably about 25 nucleotides. "Probes" as used herein refers to nucleic acid probes. These may be derived from naturally occurring or recombinant single- or double-stranded nucleic acids or may be chemically synthesized. They are useful in detecting the presence of identical or similar sequences. Such probes may be labeled with reporter molecules using nick translation, Klenow fill-in reaction, PCR or other methods well known in the art. Nucleic acid probes may be used in southern, northern, PCR based methods, hybridisation based methods or in situ hybridizations to determine whether DNA or RNA encoding a certain protein is present in a cell type, tissue, organ or fluid.
"Cells" as used herein encompass prokaryotic and/or eukaryotic cells of a defined cell line, primary cells (a cell or cell line taken directly from a living organism, which is not immortalized), cells isolated from defined tissues or cell within a tissue from different species. "Prokaryotes" as used herein refers to bacteria and archaea.
Methods for quantitative determination of nucleic acids
The labeled probe is selected so that its sequence is substantially complementary to a segment of the test locus or a reference locus. As indicated above, the nucleic acid site to which the probe binds should be located between the primer binding sites for the upstream and downstream amplification primers.
Dual lable probe technologies:
1. 5 'nuclease assays
2. Molecular beacons
3. FRET probes
4. Scorpions
The primers used in the amplification are selected so as to be capable of hybridizing to sequences at flanking regions of the locus being amplified. The primers are chosen to have at least substantial complementarity with the different strands of the nucleic acid being amplified. When a probe is utilized to detect the formation of amplification products, the primers are selected in such that they flank the probe, i.e. are located upstream and downstream of the probe.
The primer must have sufficient length so that it is capable of priming the synthesis of extension products in the presence of an agent for polymerization. The length and composition of the primer depends on many parameters, including, for example, the temperature at which the annealing reaction is conducted, proximity of the probe binding site to that of the primer, relative concentrations of the primer and probe and the particular nucleic acid composition of the probe. Typically the primer includes 15-30 nucleotides. However, the length of the primer may be more or less depending on the complexity of the primer binding site and the factors listed above. The labels used for labeling the probes or primers of the current invention and which can provide the signal corresponding to the quantity of amplification product can take a variety of forms. As indicated above with regard to the 5' fluorogenic nuclease method, a fluorescent signal is one signal which can be measured. However, measurements may also be made, for example, by monitoring radioactivity, colorimetry, absorption, magnetic parameters, or enzymatic activity. Thus, labels which can be employed include, but are not limited to, fluorophors, chromophores, radioactive isotopes, electron dense reagents, enzymes, and ligands having specific binding partners (e.g., biotin-avidin).
Monitoring changes in fluorescence is a particularly useful way to monitor the accumulation of amplification products. A number of labels useful for attachment to probes or primers are commercially available including fluorescein and various fluorescein derivatives such as, but not limited to FAM, HEX, TET and JOE; lucifer yellow, and coumarin derivatives. Labels may be attached to the probe or primer using a variety of techniques and can be attached at the 5' end, and/or the 3' end and/or at an internal nucleotide. The label can also be attached to spacer arms of various sizes which are attached to the probe or primer. These spacer arms are useful for obtaining a desired distance between multiple labels attached to the probe or primer.
In some instances, a single label may be utilized; whereas, in other instances, such as with the 5' fluorogenic nuclease assays for example, two or more labels are attached to the probe. In cases wherein the probe includes multiple labels, it is generally advisable to maintain spacing between the labels which is sufficient to permit separation of the labels during digestion of the probe through the 5'-3' nuclease activity of the nucleic acid polymerase.
In recent years, fluorescent acceptor molecules, like TAMRA, have been replaced with one or another of the growing family of dark quencher molecules. Quenchers are chemically related to fluorophores but instead of emitting absorbed fluorescence resonance energy as light they have the useful property of transforming the light energy to heat. Heat dissipation of fluorescence energy means that replacing a fluorescent acceptor like TAMRA with a quencher such as Iowa Black TM FQ will result in an oligonucleotide construct that has no measurable fluorescence as long as the oligonucleotide tether remains intact. Such constructs can greatly simplify many fluorescence assays since they do not have background fluorescence. For this reason, fluorophore - quencher dual - labeled probes have become a standard in kinetic (real-time) PCR. Quenchers absorb fluorophore emission energies over a wide range of wavelengths. This expanded dy namic range greatly adds to the utility of fluorescence quenchers, particularly in the case of multiplexing assays with different fluorophores [Integrated DNA Technologies, 2002].
Multiplexing is defined as the screening of multiple targets, the use of different reporter genes within the same experiment, or the combination of many signals into a single transmission circuit or channel [Miret et al., 2005]. Multiplexing allows for the parallel processing of multiple targets and promises to increase efficiencies, reduce costs, and improve the quality and content of screening data. In addition, it can also simplify the construction and execution of target selectivity panels. The use of multiplexing in hit discovery has been previously reported for kinases, nuclear receptors, and GPCRs. Common strategies of cell-based multiplexing assay approaches are combinations of glow- and flash-light reporter genes or reporter genes with different substrate specificity. Combinations of Firefly luciferase (Glow) with photoproteins as mtAequorin or mtClytin or Firefly luciferase (Glow) with Metridia luciferase are mixtures of both approaches [Artmann, 2008]. 5' Fluorogenic Nuclease Assays
Fluorogenic nuclease assays are a real time quantitation method that uses a probe to monitor formation of amplification product. The basis for this method of monitoring the formation of amplification product is to measure continuously PCR product accumulation using a dual-labelled fluorogenic oligonucleotide probe, an approach frequently referred to in the literature simply as the "TaqMan method".
The probe used in such assays is typically a short (about 20-25 bases) oligonucleotide that is labeled with two different fluorescent dyes. The 5' terminus of the probe is attached to a reporter dye and the 3' terminus is attached to a quenching dye, although the dyes could be attached at other locations on the probe as well. The probe is designed to have at least substantial sequence complementarity with the probe binding site. Upstream and downstream PCR primers which bind to flanking regions of the locus are added to the reaction mixture. When the probe is intact, energy transfer between the two fluorophors occurs and the quencher quenches emission from the reporter. During the extension phase of PCR, the probe is cleaved by the 5' nuclease activity of a nucleic acid polymerase such as Taq polymerase, thereby releasing the reporter from the oligonucleotide- quencher and resulting in an increase of reporter emission intensity which can be measured by an appropriate detector. Computer software is capable of recording the fluorescence intensity of reporter and quencher over the course of the amplification. The recorded values will then be used to calculate the increase in normalized reporter emission intensity on a continuous basis. The increase in emission intensity is plotted versus time, i.e., the number of amplification cycles, to produce a continuous measure of amplification. To quantify the locus in each amplification reaction, the amplification plot is examined at a point during the log phase of product accumulation. This is accomplished by assigning a fluorescence threshold intensity above background and determining the point at which each amplification plot crosses the threshold (defined as the threshold cycle number or Ct or CP). Differences in threshold cycle number are used to quantify the relative amount of PCR target contained within each tube. Assuming that each reaction functions at 100% PCR efficiency, a difference of one Ct represents a two-fold difference in the amount of starting template. The fluorescence value can be used in conjunction with a standard curve to determine the amount of amplification product present.
Probe-Based Detection Methods
These detection methods involve some alteration to the structure or conformation of a probe hybridized to the locus between the amplification primer pair. In some instances, the alteration is caused by the template-dependent extension catalyzed by a nucleic acid polymerase during the amplification process. The alteration generates a detectable signal which is an indirect measure of the amount of amplification product formed.
For example, some methods involve the degradation or digestion of the probe during the extension reaction. These methods are a consequence of the 5'-3' nuclease activity associated with some nucleic acid polymerases. Polymerases having this activity cleave mononucleotides or small oligonucleotides from an oligonucleotide probe annealed to its complementary sequence located within the locus. The 3' end of the upstream primer provides the initial binding site for the nucleic acid polymerase. As the polymerase catalyzes extension of the upstream primer and encounters the bound probe, the nucleic acid polymerase displaces a portion of the 5' end of the probe and through its nuclease activity cleaves mononucleotides or oligonucleotides from the probe. The upstream primer and the probe can be designed such that they anneal to the complementary strand in close proximity to one another. In fact, the 3' end of the upstream primer and the 5' end of the probe may abut one another. In this situation, extension of the upstream primer is not necessary in order for the nucleic acid polymerase to begin cleaving the probe. In the case in which intervening nucleotides separate the upstream primer and the probe, extension of the primer is necessary before the nucleic acid polymerase encounters the 5' end of the probe. Once contact occurs and polymerization continues, the 5'-3' exonuclease activity of the nucleic acid polymerase begins cleaving mononucleotides or oligonucleotides from the 5' end of the probe. Digestion of the probe continues until the remaining portion of the probe dissociates from the complementary strand.
In solution, the two end sections can hybridize with each other to form a hairpin loop. In this conformation, the reporter and quencher dye are in sufficiently close proximity that fluorescence from the reporter dye is effectively quenched by the quencher dye. Hybridized probe, in contrast, results in a linearized conformation in which the extent of quenching is decreased. Thus, by monitoring emission changes for the two dyes, it is possible to indirectly monitor the formation of amplification product.
Non-Probe-Based Detection Methods
A variety of options are available for measuring the amplification products as they are formed. One method utilizes labels, such as dyes, which only bind to double stranded DNA. In this type of approach, amplification product (which is double stranded) binds dye molecules in solution to form a complex. With the appropriate dyes, it is possible to distinguish between dye molecules free in solution and dye molecules bound to amplification product. For example, certain dyes fluoresce only when bound to amplification product. Examples of dyes which can be used in methods of this general type include, but are not limited to, Syber Green.TM. and Pico Green from Molecular Probes, Inc. of Eugene, Oreg., ethidium bromide, propidium iodide, chromomycin, acridine orange, Hoechst 33258, Toto-1, Yoyo-1, DAPI (4',6-diamidino-2-phenylindole hydrochloride).
Molecular beacons
Molecule beacons enable dynamic, real - time detection of nucleic acid hybridization events both in vitro and in vivo. One of the primary advantages of molecular beacons is that they can discriminate between targets that differ by as little as a single base pair change, making them ideal for investigating single nucleotide polymorphisms (SNPs). Molecular beacons are designed so that probe sequence is sandwiched between complementary sequences that form the hairpin stem. Molecular beacons must be designed so that the transition between two conformational states - the hairpin and the probe: target duplex is thermodynamically favorable. The temperature and the buffer used will influence probe specificity and must be carefully controlled. As a general rule, the melting temperature of both the hairpin structure and the probe:target duplex shou Id be 7-10 oC higher than the temperature used for detection or for primer annealing. A perfect match probe- target hybrid will be energetically more stable than the stem-loop structure whereas a mismatched probe target hybrid will be energetically less stable than the stem-loop structure. This characteristic is the basis of the extraordinary specificity offered by molecular beacons. Specificity can also be relaxed by making the probe sequence in the loop and the probe - target hybrid more stable [Integrated DNA Technologies, 2002].
Scorpions
Scorpions probes consist of a primer covalent tly linked to a spacer region followed by a probe that contains a fluorophore and a quencher. The probe contains a specific, complementary target sequence, a spacer region which forms a self - complementary stem, a fluorophore, and an internal quencher all contiguous with the primer. When not bound to the target, the probe remains in a stem - loop structure which keeps the quencher and fluorophore proximal and allows the quencher to absorb the fluorescence emitted from the fluorophore. During PCR, the primer will bind to the target and go through the first round of target synthesis. Because the primer and probe are connected, the probe will be attached to the newly synthesized target region. The spacer region prevents the DNA polymerase from copying the probe region and disrupting the stem structure. Once the second cycle begins, the probe will denature and hybridize to the target which will allow the fluorophore and quencher to be separated and the resulting fluorescence emission can be detected [Integrated DNA Technologies, 2002]. Sequencing
Methodologically sound technologies for transcriptome analysis are available and widely used since the development of microarray technology and the complete sequencing of the human genome. Formerly mRNA expression was measured by microarray techniques or real-time PCR techniques. The next-generation sequencing (NGS) methods offer high- throughput gene expression profiling, genome annotation or discovery of non-coding RNA. Sequence decoding is usually performed using dideoxy chain termination technology. With increasing importance of DNA sequencing in research and diagnostics, new methods were developed allowing a high- throughput sample treatment. The important technological developments, summarized under the term next-generation sequencing, are based on the sequencing-by-synthesis (SBS) technology called pyrosequencing. The transcriptomics variant of pyrosequencing technology is called short- read massively parallel sequencing or RNA-Seq [Stahl, 2013].
High content screening
High-content screening (HCS), also known as high-content analysis (HCA) or cellomics, is a method that is used in biological research and drug discovery to identify substances such as small molecules, peptides, or RNAi that alter the phenotype of a cell in a desired manner. Hence high content screening is a type of phenotypic screen conducted in cells. Phenotypic changes may include increases or decreases in the production of cellular products such as proteins and/or changes in the morphology (visual appearance) of the cell. High content screening includes any method used to analyze whole cells or components of cells with simultaneous readout of several parameters.
In high content screening, cells are first incubated with the substance and after a period of time, structures and molecular components of the cells are analyzed. The most common analysis involves labeling proteins with fluorescent tags, and finally changes in cell phenotype are measured using automated image analysis. Through the use of fluorescent tags with different absorption and emission maxima, it is possible to measure several different cell components in parallel. Furthermore, the imaging is able to detect changes at a subcellular level (e.g., cytoplasm vs. nucleus vs. other organelles) [Alanine, 2008; Haskins, 2004].
Binding Assays
For binding assays, the test compound is preferably a small molecule which binds to and occupies the active site of target polypeptide, thereby making the ligand binding site inaccessible to substrate such that normal biological activity is prevented. Examples of such small molecules include, but are not limited to, small peptides or peptide-like molecules. Potential ligands which bind to a polypeptide or cell include, but are not limited to, the natural ligands of known target and analogues or derivatives thereof.
In binding assays, either the test compound can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound which is bound to the target polypeptide or cell can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product. Alternatively, binding of a test compound to target polypeptide or cell can be determined without labeling either of the interactants. Determining the ability of a test compound to bind to the target or cell also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA). BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore™). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
Experiments and Figures
While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. Any reference signs should not be construed as limiting the scope. All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5'->3'.
Combination of reporter gene and transcriptome (expression) analysis readouts
Luciferases are commonly used as reporter genes in cell based assay formats for the characterization of second messenger and promotor activity modulators. These assays are used to test and characterize the activity of compounds or whole compound libraries. Often recombinant cell lines with luciferases under the control of second messenger responsive elements are used. Depending on the used luciferase the cell supernatant or cell lysate is used for activity testing. Usually the microtiter plate will be discarded after the measuring.
Surprisinly the cell lysate of the completed luminescence measurement could be used as a template lysate for a QPCR measurement without any further processing. For this the lysate or an aliquot will be transferred to a QPCR useable microtiter plate. After addition of all reagent the QPCR could be performed.
The combination of a reporter gene assay and an expression analysis readout has the following advantages:
1. Saving of time
• Usually the QPCR analysis will be done in a separate copy of the test plate, which ist leading to a doubling of time
2. Saving of resources
• The use of the same set of microtiter plates to perform the reporter gene based and the QPCR based readout is leading to a bisection of the used resources.
Enhanced assay accuracy
• The combined analysis of a particular sample with different methods and readouts results in a reduced error rate, when compared to parallel analyses conducted with samples.
Combination of data from different pathway levels
• Both readouts can be used to analyse the signaling cascade of a given target gene at different levels using the same sample, with one combinatorial assay set-up. For example, the modulation of the cAMP amount in a cell can be analysed with a reporter gene-based readout applying a cAMP responsive genetic element, whereas the activity of downstream regulated target genes may be detected by a subsequent QPCR assay within the same sample.
Multiplexed combination of data from different readouts of the identical sample
• Due to the possibility to multiplex luminescence and QPCR readouts, the combination of 2 or more luminescence with 2 or more QPCR readouts is possible. Additionally the cell lysate from one luminescence measurement is sufficient for more than one QPCR assay.
Combination of a cell-based firefly luciferase assay with QPCR
To combine a reporter gene assay (using firefly luciferase as reporter gene), a cell based assay was performed as described in example 3. The cells were incubated with increasing amounts of forskoline and incubated for the given time. After the luminescence measurement the cell lysate was used for the qualification of firefly luciferase mRNA. In Fig. 2 the relative light units for the different concentrations of forskoline are shown. The luminescence was increased with increased concentrations of forskoline. After the luminescence measurement a part of the cell lysate was transferred to a QPCR microtiter plate. The QPCR was performed as described in example 3. The relative expression was calculated as described in example 2 and is shown in Fig. 1. The relative expression of the firefly luciferase was increased with increasing concentrations of forskoline. Combination of a cell based nanoluc luciferase assay with QPCR
To combine a reporter gene assay (using nanoluc luciferase as reporter gene), a cell based assay was performed as described in example 6. The cells were incubated with increasing amounts of JQl and incubated for the given time. After the luminescence measurement the cell lysate was used for the qualification of nanoluc luciferase mRNA. In Fig. 8 the relative light units for the different concentrations of JQl are shown. The luminescence was increased with increased concentrations of JQl .
After the luminescence measurement a part of the cell lysate was transferred to a QPCR microtiter plate. The QPCR was performed as described in example 6. The relative expression was calculated as described in example 2 and is shown in Fig. 7. The relative expression of the firefly luciferase was increased with increasing concentrations of JQl .
Combination of reporter gene based assays with QPCR
Different methods for quantification or analysis of gene expression were combined with reporter gene based methods. Those methods are described, but not limited to, under "reporter genes", "expression analysis" and "multiplexing reporter gene assays". The combination of methods is independent from the used microtiter plate format and could be used, but not limited to, 96, 384, 1536 and 6144 well formats. QPCR is based on a sequence of biochemical based reactions, the efficiency and accuracy of which are greatly affected by the applied experimental conditions. All steps of the RNA quantification could be effected, for example (but not limited to): reverse transcription, DNA annealing, PCR and fluorescence detection. As an example, Magnesium from the reporter gene assays has an influence on the transcriptional analysis assay (in particular QPCR). Magnesium is a cofactor of firefly luciferase and necessary for a high sensitivity firefly luciferase based reporter gene assay [Zako, 2003]. The activity of the firefly luciferase directly depends on the Magnesium concentration in the reporter gene assay buffer (shown in Fig. 4 of [Zako, 2003]). On the other hand Magnesium is an important cofactor for PCR reactions which directly influences the accuracy with which the PCR primers hybridize with their template. The concentration of Magnesium in PCR or QPCR reaction buffers therefore is a key parameter determining the results of the methods. Consequently, the optimization of PCR or QPCR processes mainly relates to the determination of the optimal amounts of Magnesium within the reaction solutions [Roux, 2009].
To ensure a sufficient activity of the luciferase in the reporter gene assay and an optimal performance of the QPCR assay, we have developed an inventive combination of reporter gene and QPCR assay systems that surprisingly allows, without an adaption of the Magnesium concentration, a sufficient activity of the luciferase and an optimal performance of the QPCR assay.
Another example relates to the influence of detergents and salts from the reporter gene assays on transcriptional analysis assay (in particular QPCR). Luciferase is an intracellularly located enzyme. To measure the luminescence as a proportion of firefly luciferase activity it is therefore necessary to disrupt the cells in order to allow an interaction between the enzyme and its extracellularly located substrate. Cells are usually disrupted by the use of detergents. The concentration and type of detergent is cell and assay dependent [Brown, 2008; Schuck, 2003]. However, it is known in the art that the presence of detergents and the concentration of salts has a direct effect on QPCR efficacy [Shatzkes, 2014; Fig. 1]. It is known, for example, that the combined use of the detergent Igepal CA-630 with increasing concentrations of sodium results in an increase of QPCR efficacy [Shatzkes, 2014]. As further described below, the inventive combination of reporter gene and QPCR assay systems ensures an effective cell lysis whithout impairing QPCR efficacy.
Additional details, features, characteristics and advantages of the object of the invention are disclosed in the dependent claims, and the following description of the respective examples and figures, which, in an exemplary fashion, show preferred embodiments of the present invention. However, these drawings should by no means be understood as to limit the scope of the invention.
Experimental methods
Example 1 : Design of primer probes for L32, Firefly luciferase, Nanoluc luciferase
Designing of QPCR primers and probes are essential for a sensitive and target gene specific detection of mRNA . For the design of the mentioned primers and probes the Primer3 software was used, according to the instructions. For the design of primers and probes (using Primer3), the amplicon size, melting temperature, the likelihood of primer-dimer formation, the difference between primer melting temperatures, and primer location relative to particular regions of interest or those regions to be avoided were considered. The sequences of the used primers and probes are given under SEQ ID NOs 1 - 12. Example 2: QPCR measurement and calculation of relative expression
For quantitation of the mRNA of L32, firefly luciferase and nanoluc luciferase, cell lysates were transferred to 384-well or 1536-well QPCR microtiter plates. For 384-well 2 \L and for 1536-well 1 μΐ cell lysate were transferred. The preparation of the cell lysates are described in examples 3-6.
Procedure of QPCR (equipment): 384-well format:
To perform QPCR in 384-well format a LC480 (Roche) was used according to the manufacturer's specifications by using the following cycling parameters: 30 min at 48°C, followed by 10 min at 95°C, followed by 50 cycles of melting at 95°C for 15 sec and annealing/extending at 60°C for 1 min. 1536-well format:
To perform QPCR in 384well format a LC1536 (Roche) was used according to the manufacturer's specifications by using the following cycling parameters: 8 min at 50°C, followed by 8 min at 95°C, followed by 50 cycles of melting at 95°C for 15 sec and annealing/extending at 60°C for 1 mi Procedure of QPCR (reagents): 384-well format:
To perform QPCR in 384well format the Custom qPCR kit from Eurogentec was used according to the manufacturer's specifications by using the following specifications. The qPCR was performed in at total volume of 10μΕ per well with 2μΕ lysate, 1.5μΕ primer/probe mix (1.33 μΜ each), 2.5 μΕ master mix, 0.05 μΕ enzyme mix and 1.45 μΐ water
1536well format:
To perform QPCR in 1536-well format the RNA Virus Master kit from Roche was used according to the manufacturer's specifications by using the following specifications. The qPCR was performed in at total volume of 2μΕ per well with Ι μΙ. lysate, 300 nL primer/probe mix (1.33 μΜ each), 400 nL master mix, 40 nL enzyme mix and 260 nl water
Procedure of QPCR (primer probes):
To perform QPCR in 384well and 1536well format the following primer-probe combination were used to detect: a. L32
Forward primer as shown in SEQ ID NO:l, Reverse primer as shown SEQ ID NO:2, Probe as shown in SEQ ID NO: 3 b. Firefly luciferase
Forward primer as shown in SEQ ID NO:4, Reverse primer as shown in SEQ ID NO:5, Probe as shown in SEQ ID NO: 6 c. Nanoluc Luciferase
Forward primer as shown in SEQ ID NO:7, Reverse primer as shown in (SEQ ID NO:8, Probe as shown in SEQ ID NO: 9
Calculation of relative expression:
Differences in cDNA amounts in cells or lysates were corrected by normalization the expression of the gene of interest (Firefly luciferase or Nanoluc luciferase) to a housekeeping gene (L32). The relative expression was calculated as followed:
Definitons:
CP value: cycle number at detection threshold (crossing point)
CP value (as given from LC480 or LC1536 respectively) of L32: CP-L32
CP value (as given from LC480 or LCI 536 respectively) of Firefly luciferase: CP-Fire
CP value (as given from LC480 or LC1536 respectively) of Nanoluc luciferase: CP-Nano
Calculations:
Relative Expression of Firefly luciferase=2(15 (CPL32 CP Fire)) Relative Expression of Nanoluc luciferase=2(15 (CPL32 CP Nano)) Example 3: Combination of firefly luciferase with QPCR - protocol 1 in 384-well format
To combine a luminescence cell based assay with a QPCR based assay 4000 CHO cells per well were seeded on 384-well microtiter plates in 50μΙ. complete growth medium. The cells are incubated for 24 hours under standard cell culture conditions (37 °C, 5% C02). After 24h the supernatant is removed and the compound is added Buffer X in the given final concentrations. The cell are incubated for 2 hours under standard conditions. The supernant is removed and ΙΟμΙ ννεΙΙ buffer A (+ 10 U/well RNase Inhibitor) + RNase free water are added. The luminescence measuring is immediately started using a standard luminometer. The luminescence signal is measured for 60sec and integrated for 1 sec . The relative light units are shown in Fig. 2. After the decay of the luminescence signal the cell lysate could directly be used for RNA or DNA quantification or analysis. For this 2 μΐ of the lysate is transferred to a 384well QPCR microtiter plate and the prepared Master Mix is added. The cDNA synthesis is performed for 30 min at 48°C . The QPCR is performed using a LC480 (Roche) with the following protocol: 10 min at 95°C, followed by 50 cycles of melting at 95°C for 15 sec and annealing/extending at 60°C for 1 min. The relative expression is calculated as described under example 2. The relative expression of the firefly luciferase is shown in Fig. 1.
Used buffers:
Buffer A: 50mM Tris(hydroxymethyl)-aminometha (pH 8), 40mM Natrium chloride, 1.5 mM Magnesium chloride, 0.5% Octylphenoxy poly(ethyleneoxy)ethanol (IGEPAL® CA-630), 50 mM Guanidiniumthiocyanat, 23.85 mM Adenosintriphosphat, 1.215 mM Acetyl-CoA, 2.115 mM D- Luciferin
Buffer X: 2mM Calcium chloride, 20mM 2-[4-(2-hydroxyethyl)piperazin-l -yl]ethanesulfonic acid, 130mM Natrium chloride, 5mM Kalium chloride, 5mM Sodium hydrogen carbonate, 2mM Magnesium chloride hexahydrate Example 4: Combination of firefly luciferase with QPCR - protocol 2 in 384well format
To combine a luminescence cell based assay with a QPCR based assay 4000 CHO cells per well were seeded on 384well microtiter plates in 50μΙ. complete growth medium . The cells are incubated for 24 hours under standard cell culture conditions (37 °C, 5% C02). After 24h the supernatant is removed and the compound is added Buffer X in the given final concentrations. The cell are incubated for 2 hours under standard conditions. The supernant is removed and ΙΟμΙ ννεΙΙ buffer B (+ 10 U/well RNase Inhibitor) + RNase free water are added. The luminescence measuring is immediately started using a standard luminometer. The luminescence signal is measured for 60sec and integrated for 1 sec . The relative light units are shown in Fig. 2.
After the decay of the luminescence signal the cell lysate could directly be used for RNA or DNA quantification or analysis. For this 2 μΐ of the lysate is transferred to a 384well QPCR microtiter plate and the prepared Master Mix is added. The cDNA synthesis is performed for 30 min at 48°C . The QPCR is performed using a LC480 (Roche) with the following protocol: 10 min at 95°C, followed by 50 cycles of melting at 95°C for 15 sec and annealing/extending at 60°C for 1 min. .The relative expression is calculated as described under example 2. The relative expression of the firefly lucif erase is shown in Fig. 3. Used buffers:
Buffer B: 120mM N-(Tri(hydroxymethyl)methyl)glycin, 15.6 mM Magnesium chloride, 1.56 mM Ethylendiamintetraacetat, 198 mM Dithiothreitol, 31.8 mM Adenosintriphosphat, 1.62 mM Acetyl- CoA, 2.82 mM Luciferin
Assay protocol: ΙΟμΙ ννεΙΙ (+ 10 U/well RNase Inhibitor) - add RNase free water to 40μΙ. total volume
Example 5: Combination of firefly luciferase with QPCR - protocol 3 in 384well format
To combine a luminescence cell based assay with a QPCR based assay 4000 CHO cells per well were seeded on 384well microtiter plates in 50μΙ. complete growth medium. The cells are incubated for 24 hours under standard cell culture conditions (37 °C, 5% C02). After 24h the supernatant is removed and the compound is added Buffer X in the given final concentrations. The cell are incubated for 2 hours under standard conditions. The supernant is removed and ΙΟμΙ ννεΙΙ buffer C (+ 10 U/well RNase Inhibitor) + RNase free water are added. The luminescence measuring is immediately started using a standard luminometer. The luminescence signal is measured for 60sec and integrated for 1 sec . The relative light units are shown in Fig. 2. After the decay of the luminescence signal the cell lysate could directly be used for RNA or DNA quantification or analysis. For this 2 μΐ of the lysate is transferred to a 384well QPCR microtiter plate and the prepared Master Mix is added. The cDNA synthesis is performed for 30 min at 48°C . The QPCR is performed using a LC480 (Roche) with the following protocol: 10 min at 95°C, followed by 50 cycles of melting at 95°C for 15 sec and annealing/extending at 60°C for 1 min. .The relative expression is calculated as described under example 2. The relative expression of the firefly luciferase is shown in Fig. 5. Used buffers:
Buffer C: Luciferase Assay System (El 500, Promega) Assay protocol:
ΙΟμΙ ννεΙΙ (+ 10 U/well RNase Inhibitor) - add RNase free water to 40μL· total volume Example 6: Combination of nanoluc luciferase with QPCR - protocol 4 in 1536well format
To combine a luminescence cell based assay with a QPCR based assay 750 CHO cells per well were seeded on 1536-well microtiter plates in 1 L complete growth medium . The cells are incubated for 24 hours under standard cell culture conditions (37 °C, 5% C02). After 24 h Buffer X is added with compounds in the given concentrations. The cell are incubated for 6 hours under standard conditions. The supernant is removed and 1 μΐ ννεΐΐ buffer D is added. The luminescence measuring is immediately started using a standard luminometer. The luminescence signal is measured for 60 sec and integrated for 1 sec . The relative light units are shown in Fig. 2. After the decay of the luminescence signal 4 μΐ buffer E (containing 1 U/well RNase Inhibitor) is added. The cell lysate could directly be used for RNA or DNA quantification or analysis. For this 1 μΐ of the lysate is transferred to a 1536-well QPCR microtiter plate and the prepared Master Mix is added. The cDNA synthesis is performed for 30 min at 48°C . The QPCR is performed using a LC1536 (Roche) with the following protocol: 10 min at 95°C, followed by 50 cycles of melting at 95°C for 15 sec and annealing/extending at 60°C for 1 min. .The relative expression is calculated as described under example 2. The relative expression of the nanoluc luciferase is shown in Fig. 7. Used Buffers:
Buffer D: Nano-Glo® Luciferase Assay (Nl 130, Promega)
Buffer E: 1 μΐ Triton Buffer (PAA, T21-160) + 5 μΐ RNase free water
Brief Description of the Drawings
Fig. 1 : combination of firefly luciferase with QPCR - protocol 1 (expression) Fig. 2: combination of firefly luciferase with QPCR - protocol 1 (luminescence) Fig. 3 : combination of firefly luciferase with QPCR - protocol 2 (expression) Fig. 4: combination of firefly luciferase with QPCR - protocol 2 (luminescence)
Fig. 5: combination of firefly luciferase with QPCR - protocol 3 (expression) Fig. 6: combination of firefly luciferase with QPCR - protocol 3 (luminescence) Fig. 7: combination of nanoluc luciferase with QPCR - protocol 4 (expression) Fig. 8: combination of nanoluc luciferase with QPCR - protocol 4 (luminescence) Fig. 9: spectra of selected fluorescent labels
Fig. 10: absorption and fluorescnce spectrum of firefly luciferase substrate)
Fig. 1 shows the relative expression of firefly luciferase using protocol 1 as described under example 3. A: concentration Forskolin (l=dmso control, 2=0.01 μΜ, 3=0.03μΜ, 4=0.1 μΜ, 5=0.3μΜ, 6=1 μΜ, 7=3μΜ, 8=10μΜ); Β: relative expression
Fig. 2 shows the relative light units (RLU) of firefly luciferase using protocol 1 as described under example 3. A: concentration Forskolin (l=dmso control, 2=0.01 μΜ, 3=0.03μΜ, 4=0.1 μΜ, 5=0.3μΜ, 6=1 μΜ, 7=3μΜ, 8=10μΜ); Β: Total Light emitted by the luciferase-catalyzed chemiluminescent reaction, in relative light units (RLU), measured 60 seconds with integration period of 1 second
Fig. 3 shows the relative expression of firefly luciferase using protocol 2 as described under example 4. A: concentration Forskolin (l=dmso control, 2=0.01 μΜ, 3=0.03μΜ, 4=0.1 μΜ, 5=0.3μΜ, 6=1 μΜ, 7=3μΜ, 8=10μΜ); Β: relative expression Fig. 4 shows the relative light units (RLU) of firefly luciferase using protocol 1 as described under example 4. A: concentration Forskolin (l=dmso control, 2=0.01 μΜ, 3=0.03μΜ, 4=0.1 μΜ, 5=0.3μΜ, 6=1 μΜ, 7=3μΜ, 8=10μΜ); Β: Total Light emitted by the luciferase-catalyzed chemiluminescent reaction, in relative light units (RLU), measured 60 seconds with integration period of 1 second
Fig. 5 shows the relative expression of firefly luciferase using protocol 1 as described under example 5. A: concentration Actinomycin D (l=dmso control + 5μΜ Forskolin, 2=0.03 μg/mL+ 5μΜ Forskolin, 3=0.06 μg/mL+ 5μΜ Forskolin, 4=0.15 μg/mL+ 5μΜ Forskolin, 5=0.32 μg/mL+ 5μΜ Forskolin, 6=0.63 μg/mL+ 5μΜ Forskolin, 7=1.25 μg/mL+ 5μΜ Forskolin, 8=2.5 μg/mL+ 5μΜ Forskolin); relative expression
Fig. 6 shows the relative light units (RLU) of firefly luciferase using protocol 1 as described under example 5. A: concentration Actinomycin D (l=dmso control + 5μΜ Forskolin, 2=0.03 μg/mL+ 5μΜ Forskolin, 3=0.06 μg/mL+ 5μΜ Forskolin, 4=0.15 μg/mL+ 5μΜ Forskolin, 5=0.32 μg/mL+ 5μΜ Forskolin, 6=0.63 μg/mL+ 5μΜ Forskolin, 7=1.25 μg/mL+ 5μΜ Forskolin, 8=2.5 μg/mL+ 5μΜ Forskolin); B: Total Light emitted by the luciferase-catalyzed chemiluminescent reaction, in relative light units (RLU), measured 60 seconds with integration period of 1 second Fig. 7 shows the relative expression of nanoluc luciferase using protocol 4 as described under example 6. A: concentration JQ1 (l=dmso control, 2= 0.01 μΜ, 3= 0.032μΜ, 4= 0.1 μΜ, 5= 0.32μΜ, 6=1 μΜ, 7= 3.2μΜ, 8= 10μΜ); Β: relative expression
Fig. 8 shows the relative light units (RLU) of nanoluc luciferase using protocol 4 as described under example 6. A: concentration JQ1 (l=dmso control, 2= 0.01 μΜ, 3= 0.032μΜ, 4= 0.1 μΜ, 5= 0.32μΜ, 6=1 μΜ, 7= 3.2μΜ, 8= 10μΜ); Β: Total Light emitted by the luciferase-catalyzed chemiluminescent reaction, in relative light units (RLU), measured 60 seconds with integration period of 1 second Fig. 9 shows the fluorescence spectra of selected fluophores which could be used for probe labeling in QPCR. (from Fig. 1 [Van Poucke, 2012]).
Fig. 10 shows the absorption and fluorescnce spectrum of firefly luciferase substrate (from Fig. 1 [Hiyama, 2012]) Listing of Sequences
SEQ ID NO: 1 (forward primer L32)
5' GCACCAGTCAGACCGATATGT 3'
SEQ ID NO : 2 (reverse primer L32)
5' TCTCCGTAACTGTTGTCCCA 3,
SEQ ID NO:3 (probe L32)
5' AATTAAGCGTAACTGGCGGAAACCC 3'
SEQ ID NO:4 (forward primer firefly luciferase)
5 " CGTCGCCAGTCAAGTAACAA 3' SEQ ID NO: 5 (reverse primer firefly luciferase)
5 " CACCTGCTTCATGGCTTTCC 3'
SEQ ID NO: 6 (probe firefly luciferase)
S'AAAGTTGCGCGGAGGAGTTGTGTTT 3'
SEQ ID NO: 7 (forward primer nanoluc luciferase)
S'CGATCCAAAGGATTGTCCTG 3'
SEQ ID NO: 8 (reverse primer nanoluc luciferase)
5 " GGTACAGTAGGGCATAC 3'
SEQ ID NO: 9 (probe nanoluc luciferase)
5 " CGGTGAAAATGGGCTGAAGATCGA 3' SEQ ID NO: 10 (nucleotide sequence of human L32)
GACCTCCTGGGATCGCATCTGGAGAGTGCCTAGTATTCTGCCAGCTTCGGAAAGGGAG GGAAAGCAAGCCTGGCAGAGGCACCCATTCCATTCCCAGCTTGCTCCGTAGCTGGCGA TTGGAAGACACTCTGCGACAGTGTTCAGTCCCTGGGCAGGAAAGCCTCCTTCCAGGAT TCTTCCTCACCTGGGGCCGCTTCTTCCCCAAAAGGCATCATGGCCGCCCTCAGACCCCT TGTGAAGCCCAAGATCGTCAAAAAGAGAACCAAGAAGTTCATCCGGCACCAGTCAGA CCGATATGTCAAAATTAAGCGTAACTGGCGGAAACCCAGAGGCATTGACAACAGGGT TCGTAGAAGATTCAAGGGCCAGATCTTGATGCCCAACATTGGTTATGGAAGCAACAAA AAAACAAAGCACATGCTGCCCAGTGGCTTCCGGAAGTTCCTGGTCCACAACGTCAAGG AGCTGGAAGTGCTGCTGATGTGCAACAAATCTTACTGTGCCGAGATCGCTCACAATGT TTCCTCCAAGAACCGCAAAGCCATCGTGGAAAGAGCTGCCCAACTGGCCATCAGAGTC ACCAACCCCAATGCCAGGCTGCGCAGTGAAGAAAATGAGTAGGCAGCTCATGTGCAC GTTTTCTGTTTAAATAAATGTAAAAACTGCCATCTGGCATCTTCCTTCCTTGATTTTAAG TCTTCAGCTTCTTGGCCAACTTAGTTTGCCACAGAGATTGTTCTTTTGCTTAAGCCCCTT TGGAATCTCCCATTTGGAGGGGATTTGTAAAGGACACTCAGTCCTTGAACAGGGGAAT GTGGCCTCAAGTGCACAGACTAGCCTTAGTCATCTCCAGTTGAGGCTGGGTATGAGGG GTACAGACTTGGCCCTCACACCAGGTAGGTTCTGAGACACTTGAAGAAGCTTGTGGCT CCCAAGCCACAAGTAGTCATTCTTAGCCTTGCTTTTGTAAAGTTAGGTGACAAGTTATT CCATGTGATGCTTGTGAGAATTGAGAAAATATGCATGGAAATATCCAGATGAATTTCT TACACAGATTCTTACGGGATGCCTAAATTGCATCCTGTAACTTCTGTCCAAAAAGAAC AGGATGATGTACAAATTGCTCTTCCAGGTAATCCACCACGGTTAACTGGAAAAGCACT TTCAGTCTCCTATAACCCTCCCACCAGCTGCTGCTTCAGGTATAATGTTACAGCAGTTT GCCAAGGCGGGGACCTAACTGGTGACAATTGAGCCTCTTGACTGGTACTCAGAATTTA GTGACACGTGGTCCTGATTTTTTTTGGAGACGGGGTCTTGCTCTCACCCAGGCTGGGAG
Figure imgf000033_0001
CAATCCTGCTTCAGCCTCCCAAAGTACTGGGATTACAGGCATCTTCTGTAGTATATAGG TCATGAGGGATATGGGATGTGGTACTTATGAGACAGAAATGCTTACAGGATGTTTTTC TGTAACCATCCTGGTCAACTTAGCAGAAATGCTGCGCTGGGTATAATAAAGCTTTTCTA CTTCTAGTCTAGACAGGAATCTTACAGATTGTCTCCTGTTCAAAACCTAGTCATAAATA TTTATAATGCAAACTGGTCAAAAAAAAAAAAAAAAAA
SEQ ID NO: l 1 (nucleotide sequence of firefly luciferase)
GGATAGTTAAATAAGAATTATTATCAAATCATTTGTATATTAATTAAAATACTATACTG TAAATTACATTTTATTTACAATCACAGATCCGGTCATTCCGGTACTGTTGGTAAAATGG AAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCTCTAGAGGATGGAA CCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGAACAAT TGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCGGAATACTTCGAAATG TCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATCG TCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATC GGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACGTGAATTGCTCAACAGTA TGAACATTTCGCAGCCTACCGTAGTGTTTGTTTCCAAAAAGGGGTTGCAAAAAATTTTG AACGTGCAAAAAAAATTACCAATAATCCAGAAAATTATTATCATGGATTCTAAAACGG ATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTT AATGAATACGATTTTGTACCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAA TGAATTCCTCTGGATCTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCC
TGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGAT
CGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTATTTTCATT CTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGAAATTGCTT CTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAAACGCTTCCATCT TCCAGGGATACGACAAGGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTACA CCCGAGGGGGATGATAAACCGGGCGGGGTCGGTAAAGTTGTTCCATTTTTTGAAGCGA AGGTTGTGGATCTGGATACCGGGAAAACGCTGGGCGTTAATCAGAGAGGCGAATTAT GTGTCAGAGGACCTATGATTATGTCCGGTTATGTAAACAATCCGGAAGCGACCAACGC CTTGATTGACAAGGATGGATGGCTACATTCTGGAGACATAGCTTACTGGGACGAAGAC GAACACTTCTTCATAGTTGACCGCTTGAAGTCTTTAATTAAATACAAAGGATATCAGGT GGCCCCCGCTGAATTGGAATCGATATTGTTACAACACCCCAACATCTTCGACGCGGGC GTGGCAGGTCTTCCCGACGATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGA GCACGGAAAGACGATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAAC AACCGCGAAAAAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTT ACCGGAAAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGC GGAAAGTCCAAATTGTAACCTGCA
SEQ ID NO: 12 (nucleotide sequence of nanoluc luciferase)
ATGGAAGCTCGACTTCCAGCTTGGCAATCCGGTACTGTTGGTAAAGCCACCATGGTCT TCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCA AGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTC CGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCAT CATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAG GTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGT AATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATC GCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAA ATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCA ACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGTAA References
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Claims

What is claimed is:
1. A method for analysing the effects of one or more test compounds on cells or tissues, comprising at least
(i) a step comprising a cell-based assay, and
(ii) a step comprising an expression analysis related to said cells or tissues.
The method according to claim 1, wherein said cell-based assay format is based on measuring second messenger signaling or promotor activity.
The method according to any one of the aforementioned claims, wherein said cell-based assay format is a reporter gene based assay format.
4. The method according to any one of the aforementioned claims, wherein said cell-based assay format is at least one selected from the group consisting of:
• a luminescence based assay format,
• a fluorescent dye based assay format
• a FRET (Fluorescence Resonance Energy Transfer) based assay format, and/or
• a Fluorescence polarisation / anisotropy based assay format
The method according to any one of claims 1 -4, wherein the expression analysis of said cells tissues is a transcriptome analysis.
6. The method according to any one of claims 1-5, wherein the expression analysis of said cells or tissues comprises an RNA quantification.
7. The method according to any one of claims 1-6, wherein the expression analysis of said cells or tissues comprises a QPCR or RT-PCR.
8. The method according to claim 1, wherein said cell-based assay format is a luciferase based assay format, and wherein said expression analysis of said cells or tissues is a QPCR or RT- PCR.
9. The method according to any one of the aforementioned claims, wherein said cells are prokaryotic or eukaryotic cells.
10. The method according to any one of the aforementioned claims, wherein said cells are selected from the group comprising human cells, animal cells, plant cells, microbial cells, and bacterial cells.
11. The method according to any one of the aforementioned claims, wherein at least one of the steps is performed in at least one microtiter plate having a number of sample wells selected from the group consisting of 6, 24, 96, 384, 1536, 3072, 3456, 6144 and 9600 wells.
12. The method according to any one of the aforementioned claims, wherein a cell lysate or cell culture supernatant derived from the cell based assay step is subjected to the expression analysis step.
13. A method of screening a library of test compounds, which method encompasses a method according to any of the aformentioned claims.
14. Kit of parts comprising reagents suitable for performing the method according to any of claims 1-13.
15. Use of a kit of parts according to claim 14 for identifying a particular gene or gene product to be a potential target for the action of potentially therapeutic agents.
16. Use of a kit of parts according to claim 14 for screening one or more test compounds which are potentially therapeutic agents, or a library thereof, for the development of a pharmaceutical drug.
17. Use according to claim 16, wherein the potentially therapeutic agents are (i) biologies or (ii) small molecules.
18. Therapeutic agent obtained by performing the method according to any of claims 1-13.
PCT/EP2018/053810 2017-02-16 2018-02-15 Combination of reporter gene assays and transcriptional analysis WO2018149929A1 (en)

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