CN104059987A - Osteosarcoma diagnostic kit and application of CPE (carboxypeptidase E) gene to preparing osteosarcoma diagnostic kit - Google Patents
Osteosarcoma diagnostic kit and application of CPE (carboxypeptidase E) gene to preparing osteosarcoma diagnostic kit Download PDFInfo
- Publication number
- CN104059987A CN104059987A CN201410321530.6A CN201410321530A CN104059987A CN 104059987 A CN104059987 A CN 104059987A CN 201410321530 A CN201410321530 A CN 201410321530A CN 104059987 A CN104059987 A CN 104059987A
- Authority
- CN
- China
- Prior art keywords
- gene
- diagnostic kit
- osteosarcoma
- cpe
- primer sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- 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
-
- 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
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an application of a CPE (carboxypeptidase E) gene to preparing a kit for diagnosing osteosarcoma. 8 sets of satisfactory osteosarcoma cases and normal crowd genome mRNA (messenger RNA) expression chip data are taken into a research scope by utilizing NCBI (national center of biotechnology information) GEO (gene expression omnibus) data retrieval; by carrying out Meta analysis onto the chip data, the CPE gene is screened as a difference significant gene; compared with a normal structure, expression of the CPE gene in the osteosarcoma tissue is regulated up, and the conclusion is verified by using a fluorescent real-time quantification PCR (polymerase chain reaction) method. The invention further discloses a kit for diagnosing osteosarcoma and a method for diagnosing osteosarcoma by utilizing the kit. According to the application disclosed by the invention, on one hand, a novel thought is provided for researching a molecular mechanism of the osteosarcoma, and on the other hand, a more flexible diagnostic kit is provided for diagnosing osteosarcoma at an early stage.
Description
Technical field
The invention belongs to biomedicine field, relate to the application in preparation osteosarcoma diagnostic kit of a kind of osteosarcoma diagnostic kit and CPE gene.
Background technology
In transcribing group research, application is biochip technology the earliest and the most widely, first from detected sample, extract RNA, and utilize fluorescently-labeled Nucleotide that its reverse transcription is become to cDNA, through the nucleotide sequence of mark can with gene chip specific site on probe hybridization, hybridization signal and obtain cellular gene expression information after testing.Biochip technology has become a highly stable believable experimental technique, and the group data of transcribing in a large number of announcing at present mainly utilize biochip technology to produce.
Osteosarcoma is the malignant tumour that derives from mesenchymal tissue, and its tumour cell be characterized as proliferation in vivo that mainly causes a disease directly forms prematurity bone or osteoid tissue, is the modal primary malignant tumor of a kind of human skeletal's system.At present, the genes involved of domestic this disease of screening mainly adopts the method for biochip technology construction expression spectrum.Along with the development of biotechnology, experimental study has produced a large amount of microarray datas, and has been filtered out and affected gene and the signal path that development occurs osteosarcoma by gene expression chip, has identified the gene of a large amount of differential expressions.But the difference of different experiment porchs and sample causes the analytical results of each gene chip to have a lot of discordancies.
Meta analyzes and can collect the result of correlative study that same problem is delivered report, statistical integration, to obtaining more accurate or more result.This is analyzed and can produce a lot of significant difference expression genes, the incorrectness that can be avoided single research to bring.It is the powerful tool of Recognition Different expressing gene in a plurality of chip experimental studies that Meta analyzes.In addition, with Meta, analyze and can identify the differential gene that single research can not be identified, and can effectively reduce the false positive of one single chip data.
The present invention carries out Meta analysis by the osteosarcoma of having delivered being transcribed to group expression data, filter out and affect the key gene that development occurs osteosarcoma, and use the expression in fluorescence real-time quantitative PCR (QPCR) detection validation clinical sample, the present invention excavates the effect of these genes in osteosarcoma by information biology means, thereby osteosarcomatous pathogeny is probed into, for its diagnosis and treatment provide certain Research foundation.
Summary of the invention
The present invention is undertaken by the osteosarcoma of having delivered being transcribed to group expression data that Meta analyzes and QPCR verifies, finds that the expression of CPE gene in osteosarcoma tissue and the expression in healthy tissues there are differences.Compare with healthy tissues, CPE gene up-regulated in osteosarcoma tissue, by detecting the expression of CPE gene transcription level, can judge whether experimenter suffers from osteosarcoma.
The object of the present invention is to provide the purposes of CPE gene in preparation osteosarcoma diagnostic kit.Described diagnostic kit comprises SYBR Green polymerase chain reaction system, for the primer pair of increase CPE gene and house-keeping gene.SYBR Green polymerase chain reaction system comprises PCR damping fluid, dNTPs, SYBRGreen fluorescence dye.
In technique scheme, the prior art that PCR damping fluid is known to the skilled person, preferably, PCR damping fluid comprises: 25mM KCl, 2.5mM MgCl
2, 200mM (NH
4)
2sO
4.
Primer pair is that those skilled in the art can be according to the conventional design principle design of design of primers.In preferred embodiment, the forward primer sequence of amplification CPE gene is 5'-AGCATCCAGTGATAACATC-3 ', and reverse primer sequence is 5 '-CTAGGCGGTCATTCTTAC-3 '; The preferred GAPDH of house-keeping gene, the forward primer sequence of this gene that increases is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ', reverse primer sequence is 5 '-GGTGGAATCATATTGGAACA-3 '.
In a specific embodiment of the present invention, diagnostic kit of the present invention also comprises M-MLV reverse transcription system, and this reverse transcription system comprises: T repeats oligonucleotide Oligo (dT), reverse transcription reaction liquid, M-MLV reversed transcriptive enzyme, RNA enzyme inhibitors, dNTPs.
Preferably reverse transcription reaction liquid comprises: the Tris-HCL of 250mM PH8.3, the KCL of 375mM, the MgCl of 15mM
2, the DTT of 50mM.
RNA enzyme inhibitors can be selected the conventional RNA enzyme inhibitors in this area, and the noncompetitive that is preferably escherichia coli expression suppresses the recombinant protein enzyme of RNA enzyme.
In a specific embodiment of the present invention, diagnostic kit of the present invention also comprises RNA and extracts reagent, and RNA enzyme extraction pack is containing Trizol, chloroform, Virahol, 75% ethanol.
Diagnostic kit of the present invention is stored in-20 ℃, reduces multigelation as far as possible.
Another object of the present invention is to provide a kind of diagnosis osteosarcomatous test kit.Described diagnostic kit comprises SYBR Green polymerase chain reaction system, for the primer pair of increase CPE gene and house-keeping gene.Described SYBR Green polymerase chain reaction system comprises PCR damping fluid, dNTPs, SYBR Green fluorescence dye.
In technique scheme, the prior art that PCR damping fluid is known to the skilled person, preferably, PCR damping fluid comprises: 25mM KCl, 2.5mM MgCl
2, 200mM (NH
4)
2sO
4.
Primer pair is that those skilled in the art can be according to the conventional design principle design of design of primers; In preferred embodiment, the forward primer sequence of amplification CPE gene is 5'-AGCATCCAGTGATAACATC-3 ', and reverse primer sequence is 5 '-CTAGGCGGTCATTCTTAC-3 '; The preferred GAPDH of house-keeping gene, the forward primer sequence of this gene that increases is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ', reverse primer sequence is 5'-GGTGGAATCATATTGGAACA-3 '.
In a specific embodiment of the present invention, diagnostic kit of the present invention also comprises M-MLV reverse transcription system, and this reverse transcription system comprises: T repeats oligonucleotide Oligo (dT), reverse transcription reaction liquid, M-MLV reversed transcriptive enzyme, RNA enzyme inhibitors, dNTPs.
Preferably reverse transcription reaction liquid comprises: the Tris-HCL of 250mM PH8.3, the KCL of 375mM, the MgCl of 15mM
2, the DTT of 50mM.
RNA enzyme inhibitors can be selected the conventional RNA enzyme inhibitors in this area, and the noncompetitive that is preferably escherichia coli expression suppresses the recombinant protein enzyme of RNA enzyme.
In a specific embodiment of the present invention, diagnostic kit of the present invention also comprises RNA and extracts reagent, and RNA enzyme extraction pack is containing Trizol, chloroform, Virahol, 75% ethanol.
The present invention also provides a kind of diagnosis osteosarcomatous method, and described method comprises:
(1) utilize RNA to extract reagent and extract the total RNA of sample;
(2) RNA reverse transcription step (1) being obtained becomes cDNA;
(3) on fluorescence real-time quantitative PCR instrument, CPE gene and house-keeping gene are carried out to augmentation detection;
(4) by melt curve analysis analysis and electrophoresis, determine object band, Δ Δ CT method is carried out relative quantification;
(5) CPE genetic expression is raised, and shows that research object is Patients with Osteosarcoma.
The specific implementation method of step (1) is: collect after sample frozenly in liquid nitrogen, the mortar of after taking-up, tissue being put into precooling grinds, after tissue samples is powdered:
1. add Trizol, room temperature preservation 5min;
2. add chloroform 0.2ml, use forced oscillation centrifuge tube, fully mix, under room temperature, place 5min-10min;
3. after 12000rpm high speed centrifugation 15min, draw upper strata water (inhaling 70%) in another new centrifuge tube, note not being drawn onto the proteic substance between two-layer water.Move into new pipe, add isopyknic-20 ℃ pre-cold isopropanol, fully put upside down and mix, be placed in 10min on ice;
4. 12000rpm at a high speed carefully discards supernatant liquor after 15min, in the ratio of 1ml/ml Trizol, adds 75%DEPC washing with alcohol precipitation (4 ℃ of preservations), washing precipitate, and vibration mixes, 12000rpm high speed centrifugation 5min at 4 ℃;
5. discard ethanol liquid, under room temperature, place 5min fully to dry precipitation, add DEPC treated water dissolution precipitation;
6. with Nanodrop2000 ultraviolet spectrophotometer, measure RNA purity and concentration, frozen in-70 ℃.
The specific implementation method of step (2) is: with reverse transcription damping fluid, the total RNA of 1 μ g is carried out to the synthetic cDNA of reverse transcription.Adopt 25 μ l reaction systems, each sample is got the total RNA of 1 μ g as template ribonucleic acid, adds respectively following component: DEPC water in PCR pipe, 5 * reverse transcription damping fluid, 10mmol/L dNTP, 0.1mmol/l DTT, 30 μ mmol/l Oligo dT, 200U/ μ l M-MLV RT, template ribonucleic acid.Hatch 1h for 42 ℃, 72 ℃ of 10min are of short duration centrifugal.
The specific implementation method of step (3) is: adopt 25 μ l reaction systems, each sample arranges 3 parallel pipes, and all amplified reactions are all above to guarantee the reliability of result in triplicate.Prepare following reaction system: SYBR Green polymerase chain reaction system 12.5 μ l, forward primer (5 μ M/ μ l) 1 μ l, reverse primer (5 μ M/ μ l) 1 μ l, template cDNA2.0 μ l, without enzyme water 8.5 μ l; The forward primer sequence of amplification CPE gene is 5 '-AGCATCCAGTGATAACATC-3 ', and reverse primer sequence is 5 ,-CTAGGCGGTCATTCTTAC-3 '; The forward primer sequence of amplification GAPDH gene is 5'-TTTAACTCTGGTAAAGTGGATAT-3 ', and reverse primer sequence is 5 ,-GGTGGAATCATATTGGAACA-3 ', and operations is all in carrying out on ice.Amplification program is: 95 ℃ of 10min, (95 ℃ of 15s, 60 ℃ of 60s) * 45 circulations.Using SYBR Green as fluorescent marker, in the enterprising performing PCR reaction of Light Cycler quantitative real time PCR Instrument, by melt curve analysis analysis and electrophoresis, determine object band, Δ Δ CT method is carried out relative quantification.
Advantage of the present invention is with beneficial effect: it is relevant to osteosarcoma that (1) the present invention discloses CPE gene first, and CPE gene is expected to become the osteosarcomatous molecular marker of diagnosis, and provides new thinking for studying osteosarcomatous molecule mechanism.(2) utilize the mode of gene expression detection to diagnose osteosarcomatous existence whether method sensitiveer, be conducive to the early stage diagnosis of disease.
Accompanying drawing explanation:
Fig. 1 represents that fluorescence real-time quantitative PCR is determined at the relative expression quantity of CPE gene mRNA in healthy tissues and osteosarcoma tissue.
Concrete embodiment:
Below in conjunction with specific embodiment, further illustrate the present invention, embodiments of the invention are only for explaining the present invention, and do not mean that and limit the scope of the invention.
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
The embodiment 1 screening gene relevant to osteosarcoma
1.1NCBI GEO (Gene Expression Omnibus) data retrieval
GEO (Gene Expression Omnibus) database is by NCBI (U.S. state-run biotechnology information center) development and maintenance, GEO database is as the database of maximum gene expression data, this database be take chip data as main, in addition also comprise the data of some non-chip types as SAGE (serial analysis of gene expression) data, SARST (rrna sequence label is analyzed continuously) data, MS (mass spectrum) data, protein group data and high-flux sequence data of new generation (MPSS, extensive parallel sequencing technology) etc.
A. search key:
(＂osteosarcoma＂[MeSH Terms]OR osteosarcoma[All Fields])AND＂Homo sapiens＂[porgn]
B. the screening sample strategy in studying:
Restriction research type is for " expression profiling by array' ' meets the data set of following standard and will include in our research: 1. selected data collection must be complete genomic expression mRNA chip data; 2. these data come from biopsy or the cultured cells (without medicine irritation or transfected) of osteosarcoma case group and control group; 3. this research is all considered through stdn or raw data set; 4. selected data collection must comprise over 3 more than sample.Finally, there are 8 cover chip datas to collect in our research (shown in table 1).
The basic condition of the full genomic data collection of table 18 cover osteosarcoma
1.2 osteosarcoma microarray data Meta analytical resultss
By DNA chip analysis software, raw data is carried out after background correction and stdn, utilize microarray significance component software (significance analysis ofmicroarray, SAM) 8 sets of data are carried out to the screening of difference expression gene, in analytic process, set multiple and change (fold change, FC) be 1.2, false positive rate (falsediscovery rate, FDR) be≤0.05, filter out altogether 3994,27 of the gene 17s that wherein expression level raises, 3606 of the genes that expression level is lowered.
Embodiment 2QPCR checking candidate gene and osteosarcomatous relation
Result based on osteosarcoma microarray data Meta is analyzed, according to the size of P value, we select CPE gene (Meta analyzes demonstration and compares with healthy tissues, CPE gene up-regulated in osteosarcoma tissue) to verify.Collect 6 osteosarcoma tissue samples, collect 6 healthy tissues samples simultaneously, adopt fluorescence real-time quantitative PCR to carry out classical molecular biology experiment checking (qRT-PCR), concrete operation step is as follows:
(1) RNA extracts
Collect after sample frozenly in liquid nitrogen, the mortar of after taking-up, tissue being put into precooling grinds, after tissue samples is powdered:
1. add Trizol, room temperature preservation 5min;
2. add chloroform 0.2ml, use forced oscillation centrifuge tube, fully mix, under room temperature, place 5min-10min;
3. after 12000rpm high speed centrifugation 15min, draw upper strata water (inhaling 70%) in another new centrifuge tube, note not being drawn onto the proteic substance between two-layer water.Move into new pipe, add isopyknic-20 ℃ pre-cold isopropanol, fully put upside down and mix, be placed in 10min on ice;
4. 12000rpm at a high speed carefully discards supernatant liquor after 15min, in the ratio of 1ml/ml Trizol, adds 75%DEPC washing with alcohol precipitation (4 ℃ of preservations), washing precipitate, and vibration mixes, 12000rpm high speed centrifugation 5min at 4 ℃;
5. discard ethanol liquid, under room temperature, place 5min fully to dry precipitation, add DEPC treated water dissolution precipitation;
6. with Nanodrop2000 ultraviolet spectrophotometer, measure RNA purity and concentration, frozen in-70 ℃.
(2) reverse transcription
With reverse transcription damping fluid, the total RNA of 1 μ g is carried out to the synthetic cDNA of reverse transcription.Adopt 25 μ l reaction systems, each sample is got the total RNA of 1 μ g as template ribonucleic acid, adds respectively following component: DEPC water in PCR pipe, 5 * reverse transcription damping fluid, 10mmol/L dNTP, 0.1mmol/l DTT, 30 μ mmol/l Oligo (dT), 200U/ μ l M-MLV, template ribonucleic acid.Hatch 1h for 42 ℃, 72 ℃ of 10min are of short duration centrifugal.
(3) QPCR amplification check
Adopt 25 μ l reaction systems, each sample arranges 3 parallel pipes, and all amplified reactions are all above to guarantee the reliability of result in triplicate.Prepare following reaction system: SYBR Green polymerase chain reaction system 12.5 μ l, forward primer (5 μ M/ μ l) 1 μ l, reverse primer (5 μ M/ μ l) 1 μ l, template cDNA2.0 μ l, without enzyme water 8.5 μ l; The forward primer sequence of amplification CPE gene is 5 ,-AGCATCCAGTGATAACATC-3 ', and reverse primer sequence is 5 ,-CTAGGCGGTCATTCTTAC-3 '; , the forward primer sequence of amplification GAPDH gene is 5 ,-TTTAACTCTGGTAAAGTGGATAT-3 ', and reverse primer sequence is 5 ,-GGTGGAATCATATTGGAACA-3 ', operations is all in carrying out on ice.Amplification program is: 95 ℃ of 10min, (95 ℃ of 15s, 60 ℃ of 60s) * 45 circulations.Using SYBR Green as fluorescent marker, in the enterprising performing PCR reaction of Light Cycler fluorescence real-time quantitative PCR instrument, by melt curve analysis analysis and electrophoresis, determine object band, Δ Δ CT method is carried out relative quantification, result as shown in Figure 1, compare with healthy tissues, the up-regulated of CPE gene in osteosarcoma tissue, consistent with Meta analytical results.
The preparation of embodiment 3 osteosarcoma diagnostic kits
According to CPE gene and osteosarcomatous dependency, can diagnose osteosarcoma whether to occur by detecting the expression of CPE gene, the invention provides accordingly and a kind ofly based on detecting CPE genetic expression, diagnose osteosarcomatous test kit, the component in this diagnostic kit is as follows: SYBR Green polymerase chain reaction system; The primer pair of amplification CPE gene and GAPDH gene.The forward primer sequence of amplification CPE gene is 5 '-AGCATCCAGTGATAACATC-3 ', and reverse primer sequence is 5 '-CTAGGCGGTCATTCTTAC-3 '; The forward primer sequence of amplification GAPDH is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ', and reverse primer sequence is 5 ,-GGTGGAATCATATTGGAACA-3 '.SYBR Green polymerase chain reaction system comprises PCR damping fluid, dNTPs, SYBR Green fluorescence dye.PCR damping fluid composition is: 25mM KCL, 2.5mM MgCL
2, 200mM (NH
4)
2sO
4.
The preparation of embodiment 4 osteosarcoma diagnostic kits
According to CPE gene and osteosarcomatous dependency, can diagnose osteosarcoma whether to occur by detecting the expression of CPE gene, the invention provides accordingly and a kind ofly based on detecting CPE genetic expression, diagnose osteosarcomatous test kit, the component in this diagnostic kit is as follows: primer pair, the M-MLV reverse transcription system of SYBR Green polymerase chain reaction system, amplification CPE gene and GAPDH gene.The forward primer sequence of amplification CPE gene is 5 '-AGCATCCAGTGATAACATC-3 ', and reverse primer sequence is 5 '-CTAGGCGGTCATTCTTAC-3 '; The forward primer sequence of amplification GAPDH is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ', and reverse primer sequence is 5 ,-GGTGGAATCATATTGGAACA-3 '.SYBR Green polymerase chain reaction system comprises PCR damping fluid, dNTPs, SYBR Green fluorescence dye.PCR buffer composition were is: 25mM KCL, 2.5mMMgCL
2, 200mM (NH
4)
2sO
4.M-MLV reverse transcription system component is: T repeats oligonucleotide Oligo (dT), reverse transcription reaction liquid, M-MLV reversed transcriptive enzyme, RNA enzyme inhibitors, dNTPs.Reverse transcription reaction fluid component is: 250mM Tris-HCL (PH8.3), 375mM KCL, 15mM MgCL
2, 50mM DTT.RNA enzyme inhibitors is the recombinant protein enzyme that the noncompetitive of escherichia coli expression suppresses RNA enzyme.
The preparation of embodiment 5 osteosarcoma diagnostic kits
According to CPE gene and osteosarcomatous dependency, can diagnose osteosarcoma whether to occur by detecting the expression of CPE gene, the invention provides accordingly and a kind ofly based on detecting CPE genetic expression, diagnose osteosarcomatous test kit, the component in test kit is as follows: primer pair, the RNA of SYBRGreen polymerase chain reaction system, amplification CPE gene and GAPDH gene extract reagent.The forward primer sequence of amplification CPE gene is 5'-AGCATCCAGTGATAACATC-3 ', and reverse primer sequence is 5 '-CTAGGCGGTCATTCTTAC-3 '; The forward primer sequence of amplification GAPDH is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ', and reverse primer sequence is 5 ,-GGTGGAATCATATTGGAACA-3 '.SYBR Green polymerase chain reaction system comprises PCR damping fluid, dNTPs, SYBR Green fluorescence dye.PCR buffer composition were is: 25mM KCL, 2.5mM MgCL
2, 200mM (NH
4)
2sO
4.RNA extracts pack containing Trizol, chloroform, Virahol, 75% ethanol.
The preparation of embodiment 6 osteosarcoma diagnostic kits
According to CPE gene and osteosarcomatous dependency, can diagnose osteosarcoma whether to occur by detecting the expression of CPE gene, the invention provides accordingly and a kind ofly based on detecting CPE genetic expression, diagnose osteosarcomatous test kit, reagent constituents is as follows: primer pair, M-MLV reverse transcription system, the RNA of SYBR Green polymerase chain reaction system, amplification CPE gene and GAPDH gene extract reagent.The forward primer sequence of amplification CPE gene is 5'-AGCATCCAGTGATAACATC-3 ', and reverse primer sequence is 5 '-CTAGGCGGTCATTCTTAC-3 '; The forward primer sequence of amplification GAPDH is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ', and reverse primer sequence is 5 ,-GGTGGAATCATATTGGAACA-3 '.SYBR Green polymerase chain reaction system comprises PCR damping fluid, dNTPs, SYBR Green fluorescence dye.PCR buffer composition were is: 25mM KCL, 2.5mM MgCL
2, 200mM (NH
4)
28O
4.M-MLV reverse transcription system component is: T repeats oligonucleotide Oligo (dT), reverse transcription reaction liquid, M-MLV reversed transcriptive enzyme, RNA enzyme inhibitors, dNTPs.Reverse transcription reaction fluid component is: the Tris-HCL of 250mM (PH8.3), the KCL of 375mM are, the MgCL of 15mM
2, the DTT of 50mM.RNA enzyme inhibitors is the recombinant protein enzyme that the noncompetitive of escherichia coli expression suppresses RNA enzyme.RNA extracts pack containing Trizol, chloroform, Virahol, 75% ethanol.
Claims (10)
- The purposes of 1.CPE gene in preparation osteosarcoma diagnostic kit, is characterized in that, described diagnostic kit comprises SYBR Green polymerase chain reaction system, for the primer pair of increase CPE gene and house-keeping gene; Described SYBR Green polymerase chain reaction system comprises: PCR damping fluid, dNTPs, SYBRGreen fluorescence dye.
- 2. purposes according to claim 1, is characterized in that, the sequence of the primer pair of described amplification CPE gene is as follows: forward primer sequence is 5'-AGCATCCAGTGATAACATC-3 ', and reverse primer sequence is 5 ,-CTAGGCGGTCATTCTTAC-3 '; Described house-keeping gene is GAPDH, and the forward primer sequence of amplification GAPDH gene is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ', and reverse primer sequence is 5 '-GGTGGAATCATATTGGAACA-3 '.
- 3. purposes according to claim 1, is characterized in that, described PCR damping fluid comprises: 25mMKCL, 2.5mM MgCL 2, 200mM (NH 4) 2sO 4.
- 4. according to the purposes described in any one in claim 1-3, it is characterized in that, described diagnostic kit also comprises M-MLV reverse transcription system, and described reverse transcription system comprises: T repeats oligonucleotide Oligo dT, reverse transcription reaction liquid, M-MLV reversed transcriptive enzyme, RNA enzyme inhibitors, dNTPs.
- 5. purposes according to claim 4, is characterized in that, described reverse transcription reaction liquid comprises: the MgCL of the Tris-HCL of 250mM PH8.3, the KCL of 375mM, 15mM 2, 50mM DTT.
- 6. an osteosarcomatous diagnostic kit, is characterized in that, described diagnostic kit comprises SYBRGreen polymerase chain reaction system, for the primer pair of increase CPE gene and house-keeping gene; Described SYBRGreen polymerase chain reaction system comprises: PCR damping fluid, dNTPs, SYBR Green fluorescence dye.
- 7. diagnostic kit according to claim 6, it is characterized in that, the sequence of the primer pair of described amplification CPE gene is as follows: forward primer sequence is 5'-AGCATCCAGTGATAACATC-3 ', and reverse primer sequence is 5 ,-CTAGGCGGTCATTCTTAC-3 '; Described house-keeping gene is GAPDH, and the forward primer sequence of amplification GAPDH gene is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ', and reverse primer sequence is 5 '-GGTGGAATCATATTGGAACA-3 '.
- 8. diagnostic kit according to claim 6, is characterized in that, described PCR damping fluid comprises: 25mM KCL, 2.5mM MgCL 2, 200mM (NH 4) 2sO 4.
- 9. according to the diagnostic kit described in any one in claim 6-8, it is characterized in that, described diagnostic kit also comprises M-MLV reverse transcription system, and described reverse transcription system comprises: T repeats oligonucleotide Oligo dT, reverse transcription reaction liquid, M-MLV reversed transcriptive enzyme, RNA enzyme inhibitors, dNTPs.
- 10. diagnostic kit according to claim 9, is characterized in that, described reverse transcription reaction liquid comprises: the MgCL of the Tris-HCL of 250mM PH8.3, the KCL of 375mM, 15mM 2, 50mM DTT.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410321530.6A CN104059987A (en) | 2014-07-08 | 2014-07-08 | Osteosarcoma diagnostic kit and application of CPE (carboxypeptidase E) gene to preparing osteosarcoma diagnostic kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410321530.6A CN104059987A (en) | 2014-07-08 | 2014-07-08 | Osteosarcoma diagnostic kit and application of CPE (carboxypeptidase E) gene to preparing osteosarcoma diagnostic kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104059987A true CN104059987A (en) | 2014-09-24 |
Family
ID=51547917
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410321530.6A Pending CN104059987A (en) | 2014-07-08 | 2014-07-08 | Osteosarcoma diagnostic kit and application of CPE (carboxypeptidase E) gene to preparing osteosarcoma diagnostic kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104059987A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651509A (en) * | 2015-02-13 | 2015-05-27 | 北京泱深生物信息技术有限公司 | New drug target for osteosarcoma |
| CN107746882A (en) * | 2017-11-30 | 2018-03-02 | 深圳美因医学检验实验室 | A kind of fluorescence quantitative PCR detection system and its application for osteosarcoma gene screening |
-
2014
- 2014-07-08 CN CN201410321530.6A patent/CN104059987A/en active Pending
Non-Patent Citations (5)
| Title |
|---|
| ELISABETH HORING ET AL.: "The "go or grow" potential of gliomas is linked to the neuropeptide processing enzyme carboxypeptidase E and mediated by metabolic stress", 《ACTA NEUROPATHOL 》 * |
| ELISABETH HORING ET AL.: "The "go or grow" potential of gliomas is linked to the neuropeptide processing enzyme carboxypeptidase E and mediated by metabolic stress", 《ACTA NEUROPATHOL 》, 15 January 2012 (2012-01-15) * |
| GENBANK: "NCBI Reference Sequence:NP_001864.1", 《GENBANK》 * |
| RAPHAELA ET AL.: "De novo expression of EphA2 in osteosarcoma modulateds activation of the mitogenic signalling pathway", 《HISTOPATHOLOGY》 * |
| SARAVANA R.K. MURTHY ET AL.: "Carboxypeptidase E: Elevated expression correlated with tumor growth and metastasis in pheochromocytomas and other cancers", 《CELL MOL NEUROBIOL》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651509A (en) * | 2015-02-13 | 2015-05-27 | 北京泱深生物信息技术有限公司 | New drug target for osteosarcoma |
| CN104651509B (en) * | 2015-02-13 | 2016-10-19 | 北京泱深生物信息技术有限公司 | Osteosarcomatous medicine new target drone |
| CN107746882A (en) * | 2017-11-30 | 2018-03-02 | 深圳美因医学检验实验室 | A kind of fluorescence quantitative PCR detection system and its application for osteosarcoma gene screening |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhang et al. | Tumor heterogeneity and circulating tumor cells | |
| CN106350600A (en) | Application of LOC80054 in diagnosis or prognosis of pancreatic cancer | |
| CN109679957B (en) | IncRNA L NC-004208 and application of detection reagent thereof in preparation of brain glioma prognosis reagent | |
| CN108949992A (en) | A kind of biomarker relevant to esophageal squamous cell carcinoma and its classification | |
| CN107574247A (en) | A kind of the glioblastoma auxiliary diagnosis based on CLCF1 genes, prognostic evaluation kit and its application method | |
| CN108624693B (en) | MiR-577 is preparing the application in diagnosis of nephropathy marker | |
| CN103820556B (en) | A kind of pituitary tumor Disease-causing gene CCNB1 and application thereof | |
| CN109628600A (en) | A kind of biomarker for early-stage breast cancer diagnosis | |
| CN105238782A (en) | Breast tumor prognosis biomarker LncRNA detection method and clinical application thereof | |
| CN108949976A (en) | Purposes of the C12orf70 and/or C17orf107 gene in cancer of pancreas testing product | |
| CN104059987A (en) | Osteosarcoma diagnostic kit and application of CPE (carboxypeptidase E) gene to preparing osteosarcoma diagnostic kit | |
| CN107058579A (en) | Adenocarcinoma of lung related miRNA, composition and its application | |
| CN106755309A (en) | Application of the molecular marked compound in cancer of pancreas prognosis evaluation product is prepared | |
| CN105506035A (en) | Method for preparing RNA or DNA probe by taking multiple DNA fragments as formwork | |
| CN104131103B (en) | AMZ1 gene is preparing the purposes in diagnostic kit | |
| CN104630379A (en) | Non-small-cell lung cancer marker FAM107A and application thereof | |
| CN109439760B (en) | Application of ARRDC2 in evaluating development process of oral squamous cell carcinoma | |
| CN101365800B (en) | Composition and method for determination of CK19 expression | |
| CN104141013B (en) | A kind of diagnosis of colon cancer kit and preparation method thereof | |
| CN104087667B (en) | NPR3 gene is preparing the purposes in diagnostic kit | |
| CN113832232A (en) | Biomarker for predicting responsiveness of lung adenocarcinoma to platinum-containing dual-drug chemotherapy, and related product | |
| CN104131104B (en) | The purposes of RIMBP3 gene | |
| CN109593835B (en) | Method, kit and application for evaluating trace FFPE RNA sample | |
| CN108085386B (en) | The identification of the reference gene of osteosarcoma miRNA detection | |
| CN104087666A (en) | Diagnostic kit and use of PPT1 (palmitoyl-protein thioesterase 1) gene in preparation of kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20140924 |