WO2002086050A2 - Ion channel polynucleotide arrays comprising non-conserved regions identifying ion channel family member expression profiles - Google Patents
Ion channel polynucleotide arrays comprising non-conserved regions identifying ion channel family member expression profiles Download PDFInfo
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- WO2002086050A2 WO2002086050A2 PCT/DK2002/000253 DK0200253W WO02086050A2 WO 2002086050 A2 WO2002086050 A2 WO 2002086050A2 DK 0200253 W DK0200253 W DK 0200253W WO 02086050 A2 WO02086050 A2 WO 02086050A2
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Definitions
- the invention relates to ion channel arrays, methods for production of ion channel arrays, primers used in the production of ion channel arrays and kits containing ion channel arrays and further to the use of such ion channel arrays in methods for the determination of expression profiles in biological materials in which there is an interest in the expression of ion channel polynucleotides.
- arrays have a plurality of polynucleotide spots deposited on a solid surface in form of an array.
- Arrays of both polypeptides and polynucleotides have been developed and find use in a variety of applications.
- One of the applications is differential gene expression, where expression of genes in different cells or tissues (normally a control sample and a sample of the cell or tissue of interest) is compared, and any difference in the mRNA expression profile is determined.
- an array of "probe" nucleotides is contacted with a nucleic acid sample of interest such as mRNA concerted into cDNA from a particular tissue or cell.
- the object of the invention is to provide ion channel arrays, kits comprising ion channel arrays and methods to produce such ion channel arrays.
- the ion channel arrays may be used for the determination of ion channel expression profiles in biological materials and also for the identification of therapeutic, prophylactic and/or toxic agents, where the therapeutic, prophylactic and/or toxic agents directly or indirectly influence the ion channel expression profiles in biological materials.
- the invention relates to an ion channel array comprising a multiplicity of individual ion channel polynucleotide spots stably associated with a surface of a solid support, wherein an individual ion channel polynucleotide spot comprises an ion channel polynucleotide composition comprising a non-conserved region of an ion channel polynucleotide family member, the spots representing at least two different regions of an ion channel polynucleotide member of a family.
- the invention in another aspect, relates to a method of preparing an array according to the invention, said method comprising generating said non-conserved regions of ion channel polynucleotide family members, preparing a multiplicity of compositions each comprising at least a non-conserved region, and stably associating said compositions in individual spots on a surface of a solid support.
- the invention relates to a set of primers specific for non- conserved regions of ion channel polynucleotide family members, wherein the set of primers are used in the method for the production of an array according to the invention.
- the invention relates to a method for the determination of an ion channel polynucleotide expression profile in a biological material, said method comprising, obtaining a polynucleotide from the biological material, labelling said polynucleotide to obtain a labelled target polynucleotide sample, contacting at least one labelled target polynucleotide sample with an array according to the invention under conditions which are sufficient to produce a hybridisation pattern, and detecting said hybridisation pattern to obtain the ion channel polynucleotide expression profile of the biological material.
- the invention relates to a method for the determination of a difference in ion channel polynucleotide expression profiles from at least a first and a second different biological materials, said method comprising obtaining a first ion channel expression profile of the first biological material according to the method of the present invention obtaining a second ion channel expression profile of the second biological material according to the method of the present invention, comparing the first and the second ion channel expression profile to identify any differences in the ion channel expression profiles between the first and the second ion channel expression profile.
- the invention relates to a method for identifying a therapeutic, prophylactic and/or toxic agent involved in the response of ion channel polypeptides in a biological material, said method comprises obtaining a first ion channel expression profile of a first biological material according to the method of the present invention, obtaining a second ion channel expression profile of a second biological material according to the method of the present invention, treating the second biological material with a test compound; obtaining a third ion channel expression profile of the treated second biological material according to the method of the present invention, comparing the first, second and third ion channel expression profiles, and identifying any difference in the ion channel expression profile so as to identify any therapeutic, prophylactic or toxic response of the test compound on the ion channel polynucleotide in the second biological material.
- the invention relates to a diagnostic method to determine the differences of ion channel expression profiles between two different biological materials; said method comprises obtaining a first ion channel expression profile of a first biological material according to the method of the present invention, obtaining a second ion channel expression profile of a second biological material according to the method of the present invention, comparing the first and second ion channel expression profiles, and identifying any difference in the ion channel expression profile.
- the invention in a final aspect, relates to an ion channel kit for use in a hybridisation assay, said kit comprising an ion channel array according to the present invention.
- the invention provides completely novel and improved ion channel arrays, kits comprising ion channel arrays and methods to produce such ion channel arrays.
- Ion channel arrays are useful in the determination of ion channel expression profiles in biological materials and also in the identification of therapeutic, prophylactic and/or toxic agents; the therapeutic, prophylactic and/or toxic agent may directly or indirectly influence the ion channel expression profiles in biological materials.
- the present invention provides a novel ion channel array.
- the ion channel array comprises e.g. a slide onto which polynucleotide spots are applied and the polynucleotide spots represent one or more ion channel families or family members.
- the polynucleotides (or fragments of polynucleotides) spotted on the slides have been chosen in such a way that the polynucleotides have specificity for more than one species such as, e.g., humans, rats and mice, i.e.
- the polynucleotides chosen from an ion channel family member preferably have a certain degree of non-identity with other family members belonging to the same ion channel family, i.e. there is a relatively low intrafamily identity.
- an ion channel array containing polynucleotides spots with specificity for e.g. human, rat and mouse makes it possible to use the ion channel array on both human, rat and mouse derived biological material. It is therefore envisaged that the use of an ion channel array according to the present invention will lead to a better understanding of e.g. the pathogenesis of different ion related conditions or diseases in humans, since it is possible to compare biological material from humans with relevant biological material from e.g. well-known disease models in e.g. rats and mice. If an ion channel is contemplated to be involved in the pathogenesis of a human related disease (e.g.
- RNA extracted from biopsies makes it possible to confirm such a hypothesis by employing an ion channel array according to the invention.
- disease models in e.g. rats and mice are used and biological material obtained from e.g. diseased and healthy rats and/or mice is assayed by means of an ion channel array of the invention.
- biological material obtained from e.g. diseased and healthy rats and/or mice is assayed by means of an ion channel array of the invention.
- an ion channel is involved in the pathogenesis in a disease model in e.g. rat or mouse (e.g. evidenced by analysis of RNA extracted from relevant tissue) also is associated with the condition in human.
- RNA extracted from e.g. human biopsies is assayed by means of an ion channel array according to the invention and compared with the results from assays employing the relevant biological material from the diseased (and healthy) animals.
- the spotted polynucleotides on the array of the invention have a certain cross-specificity to e.g. both human, rat and mouse.
- Samples from other species e.g. pigs, dogs, chickens, cows and the like
- tissue from well known e.g. neurological as well as other disease models in mice and rate can be analysed on the ion channel array.
- polynucleotide is intended to mean a single or double stranded polymer composed of nucleotides, e.g. deoxyribonucleotides and/or ribonucleotides from about 30 to about 9,000 nucleotides in length, from about 50 to about 6,000, from about 50 to about 3,000, from about 50 to about 1 ,500, from about 50 to about 1 ,000, from about 100 to about 1 ,000, from about 200 to about 750, from about 200 to 700, from about 200 to 500 or from about 300 to about 350.
- the polynucleotides may be single or double stranded polynucleotides.
- complementarity is used in relation to the base- pairing rules of nucleotides well known for a person skilled in the art.
- Polynucleotides may be complete or partial complementary. Partial complementarity means that at least one nucleic acid base is not matched according to the base pairing rules. Complete complementarity means that all nucleotides in a polynucleotide match according to the base pairing rules. The degree of complementary between polynucleotides affects the strength of hybridisation between two polynucleotide strands.
- the inhibition by hybridisation of the complementary polynucleotide to the target polynucleotide may be analysed by techniques well known for a person skilled in the art, such as Southern blot, Northern blot, and the like under conditions of high stringency.
- a partially (substantially) homologous polynucleotide will compete for and inhibit the binding of a completely homologous sequence to the target sequence under low stringency.
- homology is intended to mean the degree of identity of one polynucleotide to another polynucleotide.
- homology is used in connection with complementarity between polynucleotides within a family or between species. There may be complete homology (i.e. 100% identity) between two or more polynucleotides.
- degree of homology may be determined by any method well known for a person skilled in the art.
- polynucleotide composition is intended to mean a composition comprising a polynucleotide together with an excipient.
- the polynucleotide compositions are applied as spots on the array.
- the ion channel polynucleotide composition comprises a non-conserved region of an ion channel polynucleotide family member.
- polynucleotide composition includes also control or calibrating compositions such as, e.g. compositions comprising polynucleotides corresponding to housekeeping genes.
- non-conserved region is intended to mean a segment of nucleotides in a polynucleotide, which compared to a segment of nucleotides in another polynucleotide has at the most about 90% identity.
- a non-conserved region of an ion channel polynucleotide family member is thus defined as a region of nucleotides corresponding to part of the polynucleotide, and the non-conserved region has less than 90% such as, e.g. less than 85% less than about 80%, less than about 75% or less than about 70% identity compared to all other polynucleotides belonging to the same ion channel polynucleotide family (intrafamily identity).
- the term "conserved region” is intended to mean a segment of nucleotides in a polynucleotide, which compared to a segment of nucleotides in another polynucleotide has more than 90%, such as at least about 92%, at least about 95% or at least about 97% identity.
- ion channel is intended to mean one or more polypeptides having the ability to transport ions across biological membranes.
- the ion channels are classified upon their ion specificity, biological function, regulation or molecular structure.
- Examples of ion channels are voltage-gated ion channels, Gap-junction ion channels, ligand-gated ion channels, heat-activated ion channels, intracellular ion channels, ion channels gated by intracellular ligands such as cyclic nucleotide-gated channels or calcium-activated ion channels, and any other polynucleotides encoding polypeptides capable of transporting ions across biological membranes.
- Ion channels are direct or indirect targets for the action of compounds, such as drugs.
- ion channel polynucleotide is intended to mean a polynucleotide encoding a polypeptide involved in transporting ions over biological membranes.
- polypeptide involved in transporting ions over biological membranes.
- polypetides there may be several polynucleotides encoding different polypetides all involved in creating the ion channel and in transporting ions across biological membranes.
- ion channel polynucleotide composition is intended to mean a polynucleotide composition, wherein the polynucleotide is a polynucleotide encoding a polypeptide involved in transporting ions over biological membranes.
- ion channel family is intended to mean a group of ion channel polypeptides, which have common characteristics such as, e.g. ion specificity (i.e. the ability to transport a specific ion species across a biological membrane with almost the same transport rate and to transport specific ion species in the same rank) and tertiary amino acid structure.
- ion specificity i.e. the ability to transport a specific ion species across a biological membrane with almost the same transport rate and to transport specific ion species in the same rank
- tertiary amino acid structure tertiary amino acid structure.
- Each family comprises individual members each having structural variations but they fulfil the requirements mentioned above with respect to being classified as a family.
- An example of an ion channel family is e.g. the voltage- dependent Cl " channels, the CIC family (see also Example 1).
- ion channel polynucleotide family is intented to mean polynucleotides encoding polypeptides of an ion channel family”.
- the polynucleotides may generally be found and downloaded from Genbank or EMBL (www.ncbi.nih.org).
- the term "intrafamily identity” is intended to mean identity within a group of members belonging to the same family.
- interspecies identity is intended to mean identity between a group of different species, such as a group comprising humans, mice and rats.
- expression profile is intended to mean the expression of the mRNAs in a biological material. While an expression profile encompasses a representation of the expression level of at least one mRNA, in practice the typical expression profile represents the expression of several mRNAs. For example, an expression profile used according to the present invention represents the expression levels of at least from about 1 to 50,000 or more different mRNAs in a biological material. The expression level of the different mRNAs is the same or different. The expression of mRNAs may be up- or down regulated resulting in different expression profiles.
- biological material includes within its meaning organisms, organs, tissues, cells or biological material produced by a cell culture.
- the biological material may be living or dead.
- the material may correspond to one or more cells from the organisms, in case the organism is a multicellular organism, the material may correspond to one or more cells from one or more tissues creating the multicellular organism.
- the biological material to be used according to the invention may be derived from particular organs or tissues of the multicellular organism, or from isolated cells obtained from a single or multicellular organism. In obtaining the sample of RNAs to be analysed from the biological material from which it is derived, the biological material may be subject to a number of different processing steps.
- Such steps might include tissue homogenisation, cell isolation and cytoplasma extraction, nucleic acid extraction and the like and such processing steps are generally well known for a person skilled in the art.
- Methods of isolating RNA from cells, tissues, organs or whole organisms are known to those skilled in the art and are described in Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbour Press) (1989).
- the biological material may be of the same kind i.e. the biological material is of the same kind of origin, such as coming from the same type of tissue, the same organism or the same type of organism or the same cell type etc.
- organism is intended to mean any single cell organism such as yeast or multicellular organism, including plants, fungi and animals, preferably mammals, such as humans, rats, pigs, cows, horses, dogs, guinea pigs, ferrets, rabbits, sheep, apes, monkeys and cats.
- target polynucleotides is intended to mean polynucleotides present in the biological material of interest.
- the target polynucleotide encodes a polypeptide, which is at least a part of an ion channel. If the target polynucleotide has a complementary polynucleotide present on the ion channel array, it will hybridise thereto and thus give rise to a detectable signal.
- non-overlapping is intended to mean that when the ion channel polynucleotide regions used in the ion channel polynucleotide composition spots are obtained from the same polynucleotide, the regions are obtained from different parts of the polynucleotide and the different parts are located in such a manner that the regions not even overlap each other by a single nucleotide.
- a polynucleotide of e.g. 1 ,000 nucleotides the regions 1-500 and 501-900 are non-overlapping.
- the non-overlapping ion channel polynucleotide regions may be located with a distance of one or more nucleotides from each other.
- primer is intended to mean a polymer of 3-50 nucleotides.
- set of primers is intended to mean one or more primers having the ability to amplify an ion channel polynucleotide region under suitable conditions.
- the length of the primers may be the same or different and dependent on the character of the ion channel polynucleotide region to be amplified. Design of such a set of primers is well known for a person skilled in the art.
- the set of primers having a sufficient length to specifically hybridise to a distinct ion channel polynucleotide in the sample and the length of the primers will be from about 3 to 50 nucleotides.
- stressed state and stressed is intended to mean that the above described “biological material” is influenced compared to the normal condition.
- the biological material may be influenced by some kind of organic/inorganic compound, an environmental agent, a drug substance, pathogen, mutagen, mitogen, receptor mediated signal or the like.
- the biological material is influenced in such a manner that the expression profile of the ion channel polynucleotides in the biological material either directly or indirectly is affected resulting in at least one difference between the expression profile of the non-stressed biological material compared to the stressed biological material.
- polynucleotides may either belong to the same family or different families and/or being polynucleotides encoding the same polypeptide from the same or different species.
- Optimal alignment of nucleotides of a polynucleotide for comparison of the homologies may be conducted using the homology algorithm (Smith and Waterman, Adv. Appl. Math. 2: 482 (1981)), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol.
- An ion channel array according to the invention has a multiplicity of individual ion channel polynucleotide spots, stably associated with a surface of a solid support.
- Each spot on the ion channel array comprises an ion channel polynucleotide composition, wherein the polynucleotide regions within the composition are of known identity, usually of known sequence, as described later on in detail.
- the ion channel polynucleotide spots may be of convenient shape but most often circular, oval or any other suitable shape.
- the ion channel polynucleotide spots may be arranged in any convenient pattern across the surface of the solid support, such as in row or columns to form a grid, in a circular pattern and the like.
- the pattern of ion channel polynucleotide spots are arranged as a grid to facilitate the evaluation of the results obtained from the analyses in which the ion channel array is used.
- the ion channel array according to the invention may be of a flexible or rigid solid support and the ion channel polynucleotide spots are stably associated thereto.
- stably associated is meant that the ion channel polynucleotide spots will be associated in their position on the solid support during the analysis in which the ion channel array is used, such as during different hybridisation, washing and detection conditions.
- the ion channel polynucleotide regions contained in the spots may be covalently or non-covalently associated to the surface of the solid support.
- the solid support to which the individual ion channel polynucleotide spots are stably associated to is made of a flexible or rigid material.
- flexible is meant that the support is capable of being bent or folded without breakage.
- rigid is meant that the support is solid and does not readily bend, i.e. the support is not flexible.
- the support may be fabricated from a variety of materials, including plastics, ceramics, metals, gels, nitrocellulose, nylon, glass and the like.
- the array may be produced according to any convenient methodology, such as preparing or obtaining the polynucleotides and then stably associate them with the surface of the support or growing them directly on the support.
- the solid support of the invention may have several configurations ranging from a simple to a more complex configuration depending on the intended use of the ion channel array.
- the size and thickness of the ion channel array is not critical as long as the ion channel array will function in the expected way and as long as the results obtained after use of the ion channel array are not changed.
- the number and amount of the ion channel polynucleotide spots is dependent on the intended use of the ion channel arrays as well as the detection system use to determine the expression profile of the biological material being evaluated by the aid of the ion channel array.
- the number of the ion channel polynucleotide spots may vary from about 2 to about 100,000 such as, e.g., from about 2 to about 50,000, from about 10 to about 25,000, from about 100 to about 10,000, from about 100 to about 5,000, from about 100 to about 1 ,000, from about 400 to about 600 or about 500 ion channel polynucleotide spots, or at least 2 such as, e.g. at least 10, at least 25, at least 50, at least 100, at least 300, at least 400, at least 500 or at least 600 spots, or even more than 100,000 spots.
- the limitations of the number of the ion channel polynucleotide spots are dependent on the way in which the evaluation of the expression profile of the biological material is performed.
- the amount of the ion channel polynucleotide regions present in the ion channel polynucleotide spot may vary and the amount will be sufficient to provide adequate hybridisation and detection of the target nucleic acid. Generally the ion channel polynucleotides will be present in each spot at a concentration corresponding to an amount of 1 pg - 100 ⁇ g or less than 100 ⁇ g of the polynucleotide. Normally, only 1 ion channel polynucleotide region is present in each spot.
- the copy number of the ion channel polynucleotide present in each ion channel polynucleotide spot will be sufficient to provide enough hybridisation for a target nucleic acid to yield a detectable signal, and generally range from about 50 fmol or less.
- An important feature of the ion channel array is i) that the majority of the ion channel polynucleotide spots represent ion channel families which have ion channel members, ii) the ion channel polynucleotide regions present in the ion channel polynucleotide spots are made up from non-conserved regions of the ion channel family members, and iii) at least two ion channel polynculeotide regions representing each ion channel family member are present on the ion channel array.
- the two or more ion channel polynucleotide regions from one ion channel family member are chosen in such a way that they are non-overlapping regions.
- ion channel polynucleotide regions on the ion channel array ensures a proper expression profile from the same ion channel polynucleotide.
- prior art arrays have suffered from the technical problem that they are not fully reliable in the sense that they produce a certain level of both false negative and false positive results.
- This technical problem has been solved with the present invention by the use of at least two regions of the same ion channel polynucleotide family member.
- Such array design greatly increases the reliability of the results generated by the array, as each determination has at least one double check or verification (positive and negative control).
- this double check may be effected in a controlled manner and to a predetermined level by selection of an appropriate number of regions of an ion channel polynucleotide family member.
- the level of double check i.e. the number of regions, may be selected specifically for the intended use and depends on several factors, which includes, but are not limited to, the length of the polynucleotide regions spotted, the degree of intra-family identity, the degree of interspecies identity, the array hybridisation conditions, the characteristics of the biological polynucleotide test sample etc.
- the number of non-conserved polypeptide regions to be chosen is also dependent inter alia on the length of the corresponding ion channel polynucleotide. Additionally, the ability to identify expression of ion channel polynucleotide which for some reason have a mutation or a deletion may increase by the use of more than one non-conserved polynucleotide region from each member of a ion channel family.
- each type of spot as defined by its content of polynucleotide region is present in a number of copies, such as 2, 3, 4, 5 or 6, in order to enhance the reliability of the results obtained in the use of the array.
- the mean value of the results obtained is calculated and used.
- the array according to the invention has several different regions of an ion channel polynucleotide family member, which may be polynucleotide regions from the same polynucleotide strand and the regions differ at least by one nucleotide.
- the non-conserved regions corresponding to a specific ion channel member of a ion channel family are preferably selected such that the selected ion channel regions have the ability to hybridise to the corresponding polynucleotide from more than one species.
- One selected ion channel region may be used for the identification of the expression profile of a certain ion channel polynucleotide in several biological materials obtained from several species such as, e.g., humans, mice and rats.
- the functionality of the ion channel array increases such that solely one type of ion channel array is needed for the evaluation of the expression profiles of ion channel polynucleotide in biological materials obtained from several species.
- One ion channel polynucleotide spot will hybridise to one single member of an ion channel family due to the selected non-conserved region of that particular ion channel.
- several different biological materials such as, e.g. material obtained from different species of animals may be used and compared for their expression profile of ion channel polynucleotides using only one type of ion channel array.
- the same strategy also applies for plants, fungi, microorganisms etc.
- Other polynucleotide spots (control spots), which may be present on the ion channel array include spots comprising genomic DNA, housekeeping genes, negative and positive control polynucleotides and the like.
- polynucleotide spots comprise polynucleotides, which are not unique, i.e they are not polynucleotide regions corresponding to ion channel polynucleotides. They are used for calibration or as control polynucleotides, and the function of these polynucleotide spots are not to give information of the expression of these polynucleotides, but rather to provide useful information, such as background or basal level of expression to verify that the analysis and the expression profiles obtained are relevant or not. Furthermore these control spots may serve as orientation spots.
- the ion channel polynucleotides of interest in the present context are those, which encode polypeptides involved in transporting ions across biological membranes.
- suitable ion channel polynucleotide families are voltage-gated ion channels, Gap-junction ion channels, ligand-gated ion channels, heat-activated ion channels, intracellular ion channels, ion channel gated by intracellular ligands such as cyclic nucleotide-gated channels or calcium-activated ion channels, and any other polynucleotides encoding polypeptides capable of transporting ions across biological membranes.
- the ion channel polynucleotides to be stably associated to the solid support may be of DNA, RNA, cDNA, natural, synthetic, semisynthetic origin or chemical analogous such as LNA or PNA.
- the ion channel polynucleotides may be obtained from one or more biological material such as an organism, an organ, a tissue and/or a cell and/or produced by a cell culture.
- the biological material may be obtained from any kind of organism, such as a microorganism, a plant, a fungus (e.g. yeast, mushrooms) or an animal.
- Examples of animals from which one or more biological material may be obtained are humans, rats, mice, pigs, cows, horses, dogs, guinea pigs, ferrets, rabbits, apes, monkeys, cats and sheep.
- the ion channel polypeptides involved in transporting ions across biological membranes may be located e.g. in an organ such as heart, liver, prostate, brain, kidney, lung etc., tissue such as nerve, muscle, connective, etc., and/or they may be found in the cells such as e.g. in the nucleus, endoplasmatic reticulum, Golgi complex, endosome, lysosome, peroxisome, mitochondtria, cytoplasm, plasma membrane, cytoskeleton.
- the length of the ion channel polynucleotides present in the ion channel polynucleotide spot is selected in such a manner that the length is sufficient to provide a strong, specific and reproducible signal, as well as a secure and tight hybridisation.
- the length will typically vary from about 3 to about 9,000 nucleotides such as, e.g., from about 3 to about 6,000, from about 3 to about 3,000, from about 10 to about 1 ,500, from about 50 to about 1 ,000, from about 100 to about 800, from about 200 to 750, from about 200 to 700, from about 200 to 500, from about 250 to 400 or preferably from about 300 to 350.
- the length of the ion channel polynucleotides present on the ion channel array is shorter than the length of the mRNA to which it corresponds.
- the ion channel polynucleotide represents a part of the full length cDNA to which it corresponds.
- the length of the ion channel polynucleotide region present in the ion channel spot is dependent on the number of polynucleotides in the selected ion channel family member.
- the non-conserved regions of ion channel polynucleotide regions contained in an ion channel polynucleotide composition may be single or double stranded non- conserved polynucleotide regions.
- the ion channel polynucleotide composition also comprises an excipient.
- Suitable excipients are solvents like e.g. water or any other aqueous medium, pH adjusting agents like buffering agents, stabilising agents, hybridising agents, coloring agents, labelling agents and the like.
- the excipients used are inert, i.e. they do not have any polynucleotide related effect.
- an important feature of the ion channel array is that the majority of the ion channel polynucleotide spots are made up from family members of ion channel polynucleotides and the ion channel polynucleotide regions present in the ion channel polynucleotide spots are made up from non-conserved regions of the ion channel polynucleotides.
- the sequences of the ion channel families may be found in GenBank (http.www.ncbi.nih.gov) and downloaded prior to sequence comparison. The sequence comparisons may be performed using any of the methods mentioned above. An example is given in Example 1 herein.
- the ion channel array preferably represents at least two different ion channel polynucleotide members of a family and/or at least two different ion channel families.
- the ion channel polynucleotide spots are made up from one ion channel polynucleotide of one ion channel polynucleotide family member.
- the ion channel polynucleotide regions present in the two ion channel spots are made up from regions of one and the same ion channel polynucleotide and the regions are at least non-overlapping with a distance of at least one nucleotide from each other.
- the ion channel polynucleotide regions are selected in such a way that they are non- conserved regions within the same ion channel family member (intrafamily) and the regions have at least 50% identity between different species (interspecies).
- an ion channel array comprising a multiplicity of individual ion channel polynucleotide spots stably associated with a surface of a solid support, wherein an individual ion channel polynucleotide spot comprises an ion channel polynucleotide composition, and the spots represent at least two different regions of an ion channel polynucleotide family member.
- the ion channel polynucleotide composition comprises a non-conserved region of an ion channel polynucleotide family member.
- the non-conserved regions of an ion channel polynucleotide family member is a stretch of nucleotides with an average length of from about 3 to about 9,000 nucleotides such as, e.g. from about 3 to about 6,000, from about 3 to about 3,000, from about 5 to 1 ,500, from about 10 to about 1 ,000, from about 50 to about 1 ,000, from about 100 to about 1 ,000, from about 200 to 750, from about 200 to 700, from about 200 to 500, from about 250 to 400 or from about 300 to 350.
- the non-conserved regions of an ion channel polynucleotide family member is a region of nucleotides which has less than 90% such as, e.g.
- intrafamily identity which means less than 90% (or, alternatively 85%, 80%, 75% or 70%) identity between polynucleotides classified as member of a specific ion channel family, see Example 1.
- the homology between members of a certain ion channel family may be determined using the methods mentioned above.
- the two or more different non-conserved polypeptide regions corresponding to one ion channel member may be identified using the same strategy and they may at least be non-overlapping regions as mentioned above.
- the non-overlapping regions may be selected from just one non-conserved region in case the polynucleotide ion channel family member contains just one non- conserved region.
- the non-conserved polypeptide regions may furthermore be selected on the basis of homology of specific regions between different species (interspecies), such as between species of microorganisms, fungi, plants or animals such as, e.g., humans, rats, mice, pigs, cows, horses, dogs, guinea pigs, ferrets, rabbits, apes, monkeys, cats and sheep.
- species such as between species of microorganisms, fungi, plants or animals such as, e.g., humans, rats, mice, pigs, cows, horses, dogs, guinea pigs, ferrets, rabbits, apes, monkeys, cats and sheep.
- the non-conserved region of the ion channel family member may have at least 50% interspecies identity such as, e.g. at least about 60%, at least about 65%, at least about 70%, at least about 75% or at least 80% interspecies identity.
- interspecies identity such as, e.g. at least about 60%, at least about 65%, at least about 70%, at least about 75% or at least 80% interspecies identity.
- At least one non-conserved polynucleotide region is present in the form of sense single-strands in a spot.
- the polynucleotides are usually present in double-stranded form, which is denatured by heating prior to contacting with the biological sample to make the sense strand available for binding with the sample polynucleotides. It is believed that in such conventional arrays a certain variable and non-controlled level of the double-stranded polynucleotides on the array does not in fact separate sufficiently to allow hybridisation with the sample polynucleotide.
- an array wherein the sense polynucleotide is present in a single-stranded form has the advantage that all strands are available for hybridisation thus resulting in an increased and more reproducible level of binding and array response.
- the said non-conserved polynucleotide region present in the form of sense single-strands in a spot is also present in the form of antisense single-strands in a separate spot.
- This embodiment involves the advantage that the spot containing the antisense strands serves as a negative control for a positive determination in the spot containing the corresponding sense strands.
- this embodiment of the invention significantly increases the reliability of the results obtained in the use of the array.
- the ion channel polynucleotide regions present in the spots preferably originate from the same species. This is preferred because it will give an optimum level of identity between the the polynucleotides of the spot on the one side and the polynucleotides of the biological material on the other side, and hence a more reliable determination.
- the ion channel array may be prepared (produced) using any convenient method and several methods are well known for a person skilled in the art, such as standard procedures according to Sambrook et al., (Molecular cloning: A laboratory manual 2 nd edition. Cold Spring Harbour Laboratory Press, New York.).
- One means of preparing the ion channel array is i) synthesising or otherwise obtaining the above mentioned non-conserved ion channel polynucleotide regions, ii) preparing the ion channel polynucleotide compositions to be used in each spot and then iii) depositing in the form of spots the polynucleotide compositions comprising the non-conserved ion channel polynucleotide regions onto the surface of the solid support, see also Examples 2-5.
- the non-conserved ion channel polynucleotide regions may be of DNA, RNA, cDNA, natural, synthetic, semisynthetic origin or chemical analogous such as LNA or PNA.
- the non-conserved regions may be obtained from any biological material such as, e.g., tissues or cells and/or produced by a cell culture.
- the biological material may be an organism, such as a microorganism, plant, fungus (e.g. yeast or mushrooms) or animal. If the organism is an animal it may be selected from a group consisting of humans, rats, mice, pigs, cows, horses, dogs, guinea pigs, ferrets, rabbits or sheep.
- the non-conserved ion channel polynucleotide regions may be prepared using any conventional methodology such as automated solid phase synthesis protocols, PCR using one or more primers specific for the non-conserved ion channel polynucleotide regions and the like. In general, PCR is advantageous in view of the large numbers of non-conserved ion channel polynucleotide regions that must be generated for each ion channel array.
- the amplified non-conserved ion channel polynucleotide regions may further be cloned in any suitable plasmid vector to enable multiplication and storage of the amplified non-conserved ion channel polynucleotide regions (see Examples 3-4).
- the prepared non-conserved ion channel polynucleotide regions may be spotted onto the solid support using any convenient methodology, including manual and automated techniques, e.g. by micro-pipette, ink jet pins etc. and any other suitable automated systems.
- An example of an automated system is the automated spotting device, Affymetrix 417.
- the ready ion channel arrays may then be stored at suitable conditions until use.
- Method for the determination of ion channel expression profiles typically means determination of the expression level of multiple mRNAs, all of them corresponding to ion channel polynucleotides.
- the detection limit of the expression level of a mRNA may be approximately 0.2 ng or less of total RNA of the biological material used to hybridise each individual ion channel polynucleotide spot.
- the expression profiles can be produced by any means known in the art, including but not limited to the methods disclosed by: Liang et al., (1992) Science 257: 967-971 ; Ivanova et al., (1995) Nucleic Acids Res 23: 2954-2958; Guilfoyl et al., (1997) Nucleic Acids Res 25(9): 1854-1858; Chee et al., (1996) Science 274: 610-614; Velculescu et al., (1995) Science 270: 484-487; Fiscker et al., (1995) Proc Natl Acad Sci USA 92(12): 5331.5335; and Kato (1995) Nucleic Acids Res 23(18): 3685-3690.
- hybridisation conditions under which the biological polynucleotide sample is contacted with the array of the invention may vary and are selected to suit the characteristics of the specific array / sample system as well as the purpose of the use of the array.
- the hybridisation conditions selected depend e.g.
- the array reaction conditions such as the type of solid support used, the type of system used for linking the ion channel polynucleotides to the solid support and the type of hybridisation chamber used; the characteristics of the biological polynucleotide test sample, such as purity, concentration, expected amount of cDNA, the quality of the cDNA etc.
- the hybridisation conditions may be adjusted to each individual array system.
- low stringent, medium stringent and high stringent hybridisation conditions are used for human samples and medium stringent samples are used for rat and mouse samples.
- an example of low stringent conditions is 40% formamide 1 M Na and a temperature of 37°C.
- An example of medium stringent conditions is 1 M Na and a temperature of 55°C.
- An example of high stringent conditions is 1 M Na and a temperature of 65°C.
- the incubation period is preferably more than 16 hours, more preferably more than 20 hours, and most preferably more than 24 hours.
- the ion channel array will be used for the evaluation of the expression profile of one or more biological materials or a mixture of biological materials.
- the method for the determination of an ion channel polynucleotide expression profile in a biological material or in a mixture of biological materials comprises obtaining a polynucleotide from the biological material(s), labelling said polynucleotide to obtain a labelled target polynucleotide sample, contacting at least one labelled target polynucleotide sample with an array as defined above under conditions which are sufficient to produce a hybridisation pattern and detecting said hybridisation pattern to obtain the ion channel polynucleotide expression profile of the biological material or the mixture of biological materials.
- the ion channel expression profile in the biological material can thus be determined to correspond to the expression of e.g. voltage-gated ion channels, Gap-junction ion channels, ligand-gated ion channels, heat-activated ion channels, intracellular ion channels, ion channel gated by intracellular ligands such as cyclic nucleotide-gated channels or calcium-activated ion channels, or more specific family members, or any other polynucleotides encoding polypeptides capable of transporting ions across biological membranes.
- the biological material or the mixture of biological materials may be in a non-stressed or a stressed stage.
- the stress may directly or indirectly influence the ion channel expression profile and thereby the polynucleotides identified which react upon that type of stress.
- the stress may be caused by a disease or a condition such as, e.g., Asthma, cystic fibrosis, chronic obstructive pulmonary disease and rhinorrhea, convulsions, vascular spasms, coronary artery spasms, renal disorders, polycystic kidney disease, bladder spasms, urinary incontinence, bladder outflow obstruction, irritable bowel syndrome, gastrointestinal dysfunction, secretory diarrhoea, ischaemia, cerebral ischaemia, ischaemic hearth disease, angina pectoris, coronary hearth disease, traumatic brain injury, psychosis, anxiety, depression, dementia, memory and attention deficits, drug addiction and/or abuse, including ***e or tobacco abuse, Parkinson's disease, Alzheimer's disease, dysmenorrhea, narcolepsy, Reynaud's disease,
- disorders associated with the neural system for example eating disorders, obsessive compulsive disorders, panic disorders, alcoholism, pain, memory deficits and anxiety. Included among these disorders are disorders such as pseudodementia or Ganser's syndrome, migraine pain, bulimia, obesity, pre-menstrual syndrome or late luteal phase syndrome, post-traumatic syndrome, memory loss, memory dysfunction, social phobia, attention deficit hyperactivity disorder, chronic fatigue syndrome, premature ejaculation, erectile difficulty, anorexia nervosa, disorders of sleep, autism, mutism, trichotillomania or mood syndrone. Auto-immune diseases, e.g.
- RNA polyA RNA
- mRNA polyA RNA
- the total RNA/mRNA can be isolated using a variety of techniques. Numerous techniques are well known (see Sambrook et al., Molecular Cloning: A Laboratory approach, Cold Spring Harbour Press, NY (1987), and Ausubel et al., Current protocols in Molecular Biology, Greene Publishing Co. NY, (1995)). In general, these techniques include a first step of lysing the cells and then a second step of enriching for or purifying RNA.
- DNA polymerase such as "reverse transcriptase” isolated from such retroviruses as
- AMV, MoMuLV or recombinantly produced are available. Many commercial sources are available (e.g. Invitrogen, Perkin Elmer, New England Biolabs, Stratagene Cloning Systems).
- the mRNA are reversed transcribed into cDNA and at the same time a label is incorporated for later detection of the hybridised amplified products on the ion channel array.
- the amplification by PCR may be performed according to Example 2.
- the label may vary dependent on the system to be used for the detection and several labels are well known in the area of molecular biology (e.g. radioactive labels, fluorescent labels, coloring labels, chemical labels etc.)
- the labelled cDNA is then denaturated and used for hybridisation on the ion channel array.
- the hybridisation conditions vary and are dependent on the aim with the expression profile obtained after the hybridisation. One example is found in
- Example 6 After hybridisation of the labelled cDNA, the ion channel array is washed to remove the cDNA, which have not hybridised to the ion channel and the hybridised labelled cDNA are detected by a suitable means and an expression profile obtained.
- two ion channel arrays will be used for the evaluation of the expression profiles in at least a first and a second biological material. The expression profiles of the first and the second biological material are compared to each other to identify any differences between the first and the second expression profile.
- the analysis comprising obtaining a first ion channel expression profile of the first biological material as described above, obtaining a second ion channel expression profile of the second biological material as described for the first biological material, comparing the first and the second ion channel expression profile to identify any differences in the ion channel expression profiles between the first and the second ion channel expression profile.
- the first and the second biological material may be of the same origin of different origins, for example two livers from the same animal species or two lungs from the same animal or from two animals of the same species etc.
- the first and the second biological material are in two different stages, i.e. the first biological material is non-stressed and the second biological material is stressed.
- the second biological material may be stressed in such as way that at least a different ion channel expression profile will be obtained. The stress may directly or indirectly influence the ion channel expression profile.
- the ion channels to be influenced by the stress of the second biological material may be voltage-gated ion channels, Gap-junction ion channels, ligand-gated ion channels, heat-activated ion channels, intracellular ion channels, ion channel gated by intracellular ligands such as cyclic nucleotide-gated channels or calcium activated ion channels.
- the ion channel expression profile of the second biological material may be directly or indirectly related to a disease, a chemical pretreatment, environmental influences or other physiological or patophysiological changes in the biological material.
- the chemical treatment may be selected from the group consisting of naturally occurring chemical entities or synthetically derived chemical entities.
- diseases or conditions that might influence the ion channel expression profile of the second biological material are those mentioned above.
- in vivo models such as, e.g., a rat model in which at least a first and a second experimental group are used.
- the first group is non-stressed and the second group stressed in such a way that the expression of one or more ion channel polynucleotides are influenced in such as way that an increase or a decrease of the expression is obtained, when the expression profiles are analysed using the ion channel array and the method according to the invention.
- the second group may be either permanently stressed or stressed during a certain period of time and after the period of stress one or more biological materials obtained from the second group and the ion channel expression profile determined
- the ion channel array is used for the evaluation of the expression profiles in at least a first and a second biological material, each material being labelled with a unique label (e.g. Cy3 and Cy5 for each sample, respectively).
- a unique label e.g. Cy3 and Cy5 for each sample, respectively.
- the ion channel array will be used for the identification of a therapeutic, prophylactic or toxic agent involved in the response of ion channel polypeptides in a biological material
- said method comprises obtaining a first ion channel expression profile of a first biological material as described above, obtaining a second ion channel expression profile of a second biological material as described above, treating the first and/or the second biological material with a test compound; obtaining a third and/or a fourth ion channel expression profile of the treated second biological material as described above, comparing the first, second, third and/or fourth ion channel expression profiles, and identifying any difference in the ion channel expression profile so as to identify any therapeutic, prophylactic or toxic response of the test compound on the ion channel polynucleotide.
- the first biological material may typically be a material in a healthy or normal condition whereas the second biological material typically may be in a diseased or not normal state.
- the first and the second biological material may have the same kind of origin.
- the first biological material may be in a non-stressed state and the second biological material may be in a stressed state and the stress may directly or indirectly influence the ion channel expression profile between the first and the second biological materials.
- the ion channel polynucleotide family which is influenced by the stress is selected from the group consisting of voltage-gated ion channels, Gap-junction ion channels, ligand-gated ion channels, heat-activated ion channels, intracellular ion channels, ion channel gated by intracellular ligands such as cyclic nucleotide-gated channels or calcium-activated ion channels, and the ion channel expression profile of the second biological material is directly or indirectly related to a disease, a chemical or biological pretreatment, environmental influences or other physiological or patophysiological changes.
- the disease may be anyone of those mentioned above.
- the test compound may be a chemical or a biological compound including therapeutic, prophylactic and/or toxic chemical entities, physiologically chemical entities, substances affecting a biological function, hormones, vitamins, nutrients, pesticides, fungicides, bacteriocides and the like.
- the method according to the third embodiment of the invention is used to identify potential therapeutic, prophylactic and/or toxic agents useful for the treatment of diseases caused by an alteration in the expression profile of the ion channel polypeptides.
- a biological model such as a rat model in which a first, second, third and/or fourth group are used.
- the first and third group is non-stressed and the second and fourth group stressed in such a way that the expression of one or more ion channel polynucleotides are influenced in such as way that an increase or a decrease of the expression is obtained.
- the third and fourth groups are treated with a test compound.
- the invention will be used in diagnostic methods to enable the determination in differences of ion channel expression profiles between two different biological material, said method comprises obtaining a first ion channel expression profile of a first biological material as described above, obtaining a second ion channel expression profile of a second biological material as described above, comparing the first and second ion channel expression profiles, and identifying any difference in the ion channel expression profile.
- the difference between the ion channel expression profile of the first and the second ion channel expression profile is directly or indirectly influenced by a disease, such as chronic pain, myotonia, multiple sclerosis, rheumatoid arthritis.
- the ion channel expression profile from more than two different biological materials are compared, such as biological materials, which are in different stages of a disease.
- the diagnostic method may be useful in the determination of diseases directly or indirectly caused by different ion channel expression profiles and by the use of such a method there will be an enhanced possibility to start the treatment of the disease at an early stage of the disease.
- the invention also relates to kits comprising the above-mentioned ion channel array.
- the kit may be used according to the above-mentioned methods for the determination of ion channel expression profiles in a biological material as defined above.
- the kit comprises the ion channel array as described above. However, the kit may further comprise reagents for generating a labelled target polynucleotide sample and/or a hybridisation buffer suitable for performing hybridisation between a biological material and the ion channel array.
- the voltage gated chloride channels in the CIC family comprise 9 members: CIC-1 , -2, -3, -4, -5, -6, -7, -Ka, and Kb (Jentsch et al. BioEssays vol. 19 No. 2, pp 117-126, 1997).
- the cDNA sequences of the member belonging to the C1C family comprise 9 members: CIC-1 , -2, -3, -4, -5, -6, -7, -Ka, and Kb (Jentsch et al. BioEssays vol. 19 No. 2, pp 117-126, 1997).
- the numbering of the nucleotide sequence start at the 5'-end of the polynucleotide and correspond to A in the startcodon (ATG). Thereafter the ratCIC-2 (ace. No. NM_017137) and mouseCIC-2 cDNA (Ace. No. NM_009900) sequence downloaded from Genbank and compared with the human CIC-2 cDNA (AF026004) nucleotide sequence and analysed to identify homologous regions using clustal alignment.
- nucleotide sequence 1 -350 was found to have 88% homology (interspecies) and the region corresponding to the nucleotide sequence 649-1314 was found to have 91% homology (interspecies) in between the human CIC, rat CIC-2 and mouse CIC-2.
- EXAMPLE 2 The region corresponding to nucleotide sequence 1 -350 was found to have 88% homology (interspecies) and the region corresponding to the nucleotide sequence 649-1314 was found to have 91% homology (interspecies) in between the human CIC, rat CIC-2 and mouse CIC-2.
- the two regions 1-350 and 649-1314 were amplified using conventional PCR well known for a person skilled in the art using following primers:
- RNA Human kidney total RNA was purchased from Life technology (11410-016) Recombinant plasmids are used as templates when cDNA encoding a given ion channel previously have been cloned.
- the cDNA was synthesized using the Omniscript RT Kit (Quigen, 205111). The amplification of the two regions were performed using:
- PCR generated fragments were separated using on a conventional agarose gel and cloned into the pCRII-TOPO vector according to the TOPO TA cloning kit (Invitrogene) and the nucleotide sequence was analysed using CEQ 2000 DNA Analysis System (Beckman Coulter, U.S.A.).
- the glycerol stocks were prepared in 96 wells-trays (Corning Cat. No. cci3793) on a Biomek. 50 ⁇ l glycerol media (38% glycerol, 2 x LB) was transferred into each well of a plane 96-well tray (Corning Cat. No. cci3793). Then, 50 ⁇ l bacterial culture was transferred into each well of the plane 96-well tray and mixed with the glucerol media. A Storage Mat-I lid (Corning, Cat. No. 3094) was placed on each tray and the trays stored at -80°C.
- Circlegrow medium (obtained from Bio101 , Carlsbad, CA 92008, U.S.A.). Circlegrow medium/ampicillin was added to each well in a 4 x 2 ml 96 deep well-tray (Corning Cat.No. cci 3961). The appropriate bacterial glycerol stock was added to each well. The tray was sealed with sealing sheet (Merck Eurolab A/S, Denmark), and incubated with shaking at 37°C for 16 hours prior to plasmid purification. The plasmid purification was performed using Biomek or a conventional method according to Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbour Press) (1989) well known for a person skilled in the art.
- Example 4 The plasmids obtained in Example 4 were subjected to PCR using flanking primers. The resulting PCR product was spotted onto a 3D-Link Amine-Binding slide (array). The PCR reaction and spotting were carried out using standard methods as described e.g. in "Microarray Biochip Technology" by Mark Schena and "DNA Microarrays: A Practical Approach” by Mark Schena.
- 3D-Link Amine-Binding slide may be obtained from Motorola Inc. 2501 San Pedro N. E. Suite 202 Albuquerque, NM 87110 United States.
- each of the arrays corners are marked by a double labelled Cy3 and Cy5 primer with a 5' end amino group (5'-Cy3/5) in a final concentration of 1 pmol/ ⁇ l to enable the possibility to place a grid on the scanned array.
- a double labelled Cy3 and Cy5 primer with a 5' end amino group (5'-Cy3/5) in a final concentration of 1 pmol/ ⁇ l to enable the possibility to place a grid on the scanned array.
- seal the fragments with sealing tape and store at -20°C until use.
- 3MM paper is pre-wetted with saturated NaCl solution. Place all the slides in a slide box without a lid. Place the slide boxes in a plastic bag containing the NaCl saturated 3MM paper. Close the plastic bag and incubate the slides. After incubation, remove the slides from the plastic bag. Store at room temperature until blocking of free reactive groups on the slides.
- RNA is obtained according to Example 2. Precipitate the RNA by centrifugation and wash the RNA pellet in 70% ethanol. Precipitate the RNA at 15,000 x g for 15 min. Discard the supernatant and let the pellet air-dry. Adjust the RNA concentration to 1 ⁇ g/ ⁇ l with DEPC-H 2 0. In 2 separate tubes add 25 ⁇ l total RNA (1 ⁇ g/ ⁇ l) and 7 ⁇ l DEPC treated H 2 0. Add 4 ⁇ l of oligo-dT (e.g. T25V primer) (1 ⁇ g/ ⁇ l) to each tube. Incubate the tubes in a Thermal cycler at 65°C for 3 min.
- oligo-dT e.g. T25V primer
- Probe-Mix Denature the Probe-Mix at 96°C for 3 min. Transfer the Probe-Mix directly to 55°C for 30 sec. Place the Probe-Mix on ice. Add the Probe-Mix to the array slide. Place the slide in a box and inside the petri dish with the pre-wetted 3MM paper. Replace the lid back onto the petri dish. Place the petri dish in a plastic bag.
- the scanning of the slide and evaluation were performed using Affymetrix 418 Scanner, Affymetrix 418 Scanner Software and the software ImaGene 4.0 (BioDiscovery) according to the user manual.
- regions having the lowest existing sequence identity are used.
- the regions identified on the basis of a low intrafamily sequence identity are compared to the corresponding ion channel family members from other species to determine the level of sequence identity.
- the level of interspecies identity varies from family to family and from family member to family member, but in general it is initially attempted to obtain regions having a level of identity of above 75%. If this condition is not met, a different region is selected on the basis of the intrafamily identity as described above, and the interspecies identity of the new region is determined. This procedure is repeated a number of times to optimise the region selected with respect to partly intrafamily identity and partly interspecies identity.
- Table 1 shows for each of the 233 regions identified the ion channel family member and region, the Genbank Accession number, whether it is included in the array, the intrafamily identity and the interfamily identity.
- the ion channel families are grouped together and separated by a blank row.
- KCNQ calcium-activated chloride channel
- KCNE beta subunit of voltage-dependent, non-inactivating K-channel, subtype Q
- Eag voltage-dependent K-channel, subtype Eag (ether-a-go-go)
- Erg voltage-dependent K-channel, subtype Erg (ether-a-go-go-related gene)
- GABA GABA(gamma aminobutyric acid)-gated chloride channel
- K-channelGluR AMPA-sensitive glutamate- gated cation channel
- KA Kanaite sensitive, glutamate-gated cation channel
- NR N-methyl-D-aspartate-sensitive, glutamate-gated cation channel
- TRPC transient-receptor-potential-gated cation channel
- subfamily C TRPV: transient-receptor-potential-gated cation channel
- subfamily V V
- TRPM transient-receptor-potential-gated cation channel, subfamily M
- Table 2 shows the primers used for the PCR amplification of three regions identified.
- two of the spots of the array produced in Example 7, viz. two IK ion channel family member regions, serve as control spots used to validate the functionality of the array.
- the array is contacted with a) a polynucleotide sample obtained from a biological material of non-transfected HEK293 cells and b) a polynucleotide sample obtained from a biological material of HEK293 cells transfected with a vector comprising the polynucleotide encoding the IK ion channel.
- the polynucleotide samples were prepared and processed as described in Example 6.
- DF means detection factor and expresses the amount of sample polynucleotide bound to a spot. A value of above about 4 indicates that the amount of polynucleotide in the sample has been sufficient to give a reliable measurement.
- the mean ratio is the mean ratio of the measurements of sample a) to the measurements of sample b) expressing the level of polynucleotide upregulation.
- Each spot is present in duplicate, and the measurements and DF's are given in for both spots in a duplicate pair. As will appear from the results, for both control spots the response is strongly upregulated for the sample originating from the transfected cells and the response of the sample originating from the non-transfected cells is approximately zero.
- This Example describes the embodiment of the invention, wherein the ion channel family member polynucleotide region is present as sense and anti-sense single strands in separate spots.
- the array described in Examples 7 and 8 was supplemented with four additional spots comprising sense and antisense single strands for the polynucleotide regions HulK 154-1284 and HulK 154-777, cf. spot 4 and 5 of Table 1.
- the array is contacted with a) a polynucleotide sample obtained from a biological material of non- transfected HEK293 cells and b) a polynucleotide sample obtained from a biological material of HEK293 cells transfected with a vector comprising the polynucleotide encoding the IK ion channel family member.
- the polynucleotide samples were prepared and processed as described in Example 6. Table 4 shows the results.
- the response of the antisense spots is approximately zero indicating no binding of sample polynucleotide.
- the response of the sense spots is strong and the upregulation for the sample originating from the transfected cells in relation to the non-transfected cells is higher for the sense spots than for the corresponding spots containing double-stranded polynucleotides.
- the detection factors for the sense spots are much higher than the detection factors for the corresponding spots containing double-stranded polynucleotides indicating a higher level of binding of sample polynucleotide.
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EP02732428A EP1425377A2 (en) | 2001-04-20 | 2002-04-18 | Ion channel arrays |
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WO1999005276A1 (en) * | 1997-07-25 | 1999-02-04 | Zymogenetics, Inc. | Human chloride ion channel zsig44 |
WO1999057324A1 (en) * | 1998-05-06 | 1999-11-11 | Case Western Reserve University | Analysis of gene family expression |
WO2002029116A2 (en) * | 2000-10-06 | 2002-04-11 | Ortho-Mcneil Pharmaceutical, Inc. | Gene expression profiling of antidepressant action in the brain |
WO2002040541A2 (en) * | 2000-10-27 | 2002-05-23 | Incyte Genomics, Inc. | Transporters and ion channels |
WO2002077237A2 (en) * | 2001-02-09 | 2002-10-03 | Incyte Genomics, Inc. | Transporters and ion channels |
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US6492505B1 (en) * | 1999-02-01 | 2002-12-10 | Incyte Genomics, Inc. | Composition for detection of genes encoding membrane-associated proteins |
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Also Published As
Publication number | Publication date |
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EP1425377A2 (en) | 2004-06-09 |
WO2002086050A3 (en) | 2004-03-25 |
AU2002304907A1 (en) | 2002-11-05 |
US20040118060A1 (en) | 2004-06-24 |
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