CN104087666B - A kind of purposes of diagnostic kit and PPT1 genes in its preparation - Google Patents

A kind of purposes of diagnostic kit and PPT1 genes in its preparation Download PDF

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CN104087666B
CN104087666B CN201410321626.2A CN201410321626A CN104087666B CN 104087666 B CN104087666 B CN 104087666B CN 201410321626 A CN201410321626 A CN 201410321626A CN 104087666 B CN104087666 B CN 104087666B
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genes
ppt1
osteosarcoma
osteosarcomatous
diagnostic kit
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CN104087666A (en
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杨祚璋
张娅
陈彦锦
刘雪峰
许达
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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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Abstract

The invention discloses purposes of the PPT1 genes in the osteosarcomatous test kit of diagnosis is prepared.Satisfactory 8 sets of osteosarcoma cases and normal population genome mRNA expression chip data are included research range using NCBI GEO data retrievaies by the present invention, by carrying out Meta analyses to said chip data, it is significant difference gene to filter out PPT1 genes, compare normal structure, PPT1 genes up-regulated in osteosarcoma tissue, and use the above-mentioned conclusion of fluorescence real-time quantitative PCR method validation.Accordingly, the invention also discloses a kind of diagnose osteosarcomatous test kit and diagnose osteosarcomatous method using the test kit.One aspect of the present invention provides new thinking to study osteosarcomatous molecule mechanism, on the other hand provides more sensitive diagnostic kit for early diagnosiss osteosarcoma.

Description

A kind of purposes of diagnostic kit and PPT1 genes in its preparation
Technical field
The invention belongs to biomedicine field, is related to a kind of osteosarcoma diagnostic kit and PPT1 genes and is preparing osteosarcoma Application in diagnostic kit.
Background technology
Osteosarcoma is derived from the malignant tumor of mesenchymal tissue, and its principal causative is characterized as that the tumor cell of proliferation in vivo is straight Connect to form immaturity bone or osteoid tissue, be a kind of modal primary malignant tumor of human skeletal system.At present, domestic sieve The method for selecting the related gene of the disease mainly to use biochip technology construction expression spectrum.With sending out for biotechnology Exhibition, experimentation generate substantial amounts of microarray data, and have filtered out the generation of impact osteosarcoma by gene expression chip The gene and signal path of development, has identified the gene of a large amount of differential expressions.But the difference of different experiment porch and sample There are many discordances in the different analysis result for causing each gene chip.
Meta analyses can be collected to the result of the delivered correlational study report of same problem, statistical integration, To obtaining more accurate or more results.This analysis can produce many significant difference expression genes, be avoided that single grinding Study carefully the incorrectness brought.Meta analyses are the powerful tools of the Recognition Different expressing gene in the research of multiple array experiments. Additionally, the differential gene that single research can not be recognized is can recognize that with Meta analyses, and can be effectively reduced one single chip number According to false positive.
The present invention expresses data by the osteosarcoma transcript profile to having delivered and carries out Meta analyses, filters out impact osteosarcoma There is the key gene of development, and the expression in checking clinical sample, the present invention are detected using fluorescence real-time quantitative PCR (QPCR) Effect of these genes in osteosarcoma is excavated by bioinformatics means, so as to visit to osteosarcomatous pathogeny Study carefully, be that its diagnosis and treatment provide certain Research foundation.
The content of the invention
The present invention expresses data by the osteosarcoma transcript profile to having delivered and carries out Meta analyses and QPCR checkings, finds Expression of the PPT1 genes in osteosarcoma tissue is had differences with expression in the normal tissue.Compared with normal structure, PPT1 Gene up-regulated in osteosarcoma tissue, by the expression for detecting PPT1 gene transcription levels, it can be determined that experimenter Whether osteosarcoma is suffered from.
It is an object of the invention to provide purposes of the PPT1 genes in osteosarcoma diagnostic kit is prepared.The diagnosis examination Agent box includes SYBR Green polymerase chain reactions system, the primer pair for expanding PPT1 genes and house-keeping gene.SYBR Green polymerase chain reactions system includes PCR buffer, dNTPs, SYBR Green fluorescent dyes.
In above-mentioned technical proposal, the prior art that PCR buffer is known to the skilled person, it is preferable that PCR is buffered Liquid is included：25mM KCl, 2.5mM MgCl2, 200mM (NH4)2SO4。
Primer pair is that those skilled in the art can be designed according to the conventional design principle of design of primers.Preferred embodiment party In case, the forward primer sequence for expanding PPT1 genes is 5 '-ATGGTGTCAGATGAATATG-3 ', reverse primer sequences are 5 '- CATGTACTTATTGGCACAA-3’；The preferred GAPDH of house-keeping gene, expand the gene forward primer sequence be 5 '- TTTAACTCTGGTAAAGTGGATAT-3 ', reverse primer sequences are 5 '-GGTGGAATCATATTGGAACA-3 '.
In a specific embodiment of the present invention, the diagnostic kit of the present invention also includes M-MLV reverse transcription bodies System, the reverse transcription system include：T repeats oligonucleotide Oligo (dT), reverse transcription reaction liquid, M-MLV reverse transcriptases, RNase suppression Preparation, dNTPs.
It is preferred that reverse transcription reaction liquid is included：The MgCl of the KCL of the Tris-HCL of 250mM PH8.3,375mM, 15mM2, The DTT of 50mM.
RNase inhibitor can select RNase inhibitor commonly used in the art, the preferably noncompetitive of escherichia coli expression Suppress the recombinant protease of RNase.
In a specific embodiment of the present invention, the diagnostic kit of the present invention also includes RNA extracts reagents, RNase extracts reagent includes Trizol, chloroform, isopropanol, 75% ethanol.
The diagnostic kit of the present invention is stored in -20 DEG C, reduces multigelation as far as possible.
A further object of the present invention is to provide a kind of diagnosis osteosarcomatous test kit.The diagnostic kit is included SYBR Green polymerase chain reactions system, the primer pair for expanding PPT1 genes and house-keeping gene.The SYBR Green polymerase chain reactions system includes PCR buffer, dNTPs, SYBR Green fluorescent dyes.
In above-mentioned technical proposal, the prior art that PCR buffer is known to the skilled person, it is preferable that PCR is buffered Liquid is included：25mM KCl, 2.5mM MgCl2, 200mM (NH4)2SO4。
Primer pair is that those skilled in the art can be designed according to the conventional design principle of design of primers；Preferred embodiment party In case, the forward primer sequence for expanding PPT1 genes is 5 '-ATGGTGTCAGATGAATATG-3 ', reverse primer sequences are 5 '- CATGTACTTATTGGCACAA-3’；The preferred GAPDH of house-keeping gene, expand the gene forward primer sequence be 5 '- TTTAACTCTGGTAAAGTGGATAT-3 ', reverse primer sequences are 5 '-GGTGGAATCATATTGGAACA-3 '.
In a specific embodiment of the present invention, the diagnostic kit of the present invention also includes M-MLV reverse transcription bodies System, the reverse transcription system include：T repeats oligonucleotide Oligo (dT), reverse transcription reaction liquid, M-MLV reverse transcriptases, RNase suppression Preparation, dNTPs.
It is preferred that reverse transcription reaction liquid is included：The MgCl of the KCL of the Tris-HCL of 250mM PHS.3,375mM, 15mM2, The DTT of 50mM.
RNase inhibitor can select RNase inhibitor commonly used in the art, the preferably noncompetitive of escherichia coli expression Suppress the recombinant protease of RNase.
In a specific embodiment of the present invention, the diagnostic kit of the present invention also includes RNA extracts reagents, RNase extracts reagent includes Trizol, chloroform, isopropanol, 75% ethanol.
Present invention also offers diagnosing osteosarcomatous method, methods described includes：
(1) sample total serum IgE is extracted using RNA extracts reagents；
(2) the RNA reverse transcriptions for obtaining step (1) are into cDNA；
(3) PPT1 genes and house-keeping gene are carried out into augmentation detection on fluorescence real-time quantitative PCR instrument；
(4) purpose band is determined by melt curve analysis analysis and electrophoresis, Δ Δ CT methods carry out relative quantification；
(5) PPT1 gene expressions are raised, and show that object of study is Patients with Osteosarcoma.
The specific implementation method of step (1) is：It is frozen after collecting sample that tissue is put into pre-cooling after liquid nitrogen, taking-up It is ground in mortar, after tissue samples are powdered：
1. Trizol, room temperature preservation 5min are added；
2. the imitative 0.2ml of chlorination, uses forced oscillation centrifuge tube, fully mixes, places 5min-10min under room temperature；
3. draw upper strata aqueous phase (inhaling 70%) in another new centrifuge tube, to note not after 12000rpm high speed centrifugations 15min The protein substance between two-layer water phase is drawn onto.New pipe is moved into, isopyknic -20 DEG C of pre- cold isopropanols are added, it is fully reverse mixed It is even, it is placed in 10min on ice；
4. 12000rpm carefully discards supernatant at a high speed after 15min, adds 75% in the ratio of 1ml/ml Trizol DEPC washing with alcohol precipitation (4 DEG C of preservations), washing precipitate, vibration mixing, 12000rpm high speed centrifugations 5min at 4 DEG C；
5. ethanol liquid is discarded, places 5min fully to dry precipitation under room temperature, add the water dissolution that DEPC was processed to sink Form sediment；
6. RNA purity and concentration are measured with Nanodrop2000 ultraviolet spectrophotometers, it is frozen in -70 DEG C.
The specific implementation method of step (2) is：Reverse transcription synthesis cDNA is carried out with RT Buffer to 1 μ g total serum IgEs.Adopt With 25 μ l reaction systems, each sample takes 1 μ g total serum IgEs as template ribonucleic acid, following components is separately added in PCR pipe：DEPC Water, 5 × RT Buffer, 10mmol/L dNTP, 0.1mmol/1DTT, 30 μm of mol/1Oligo dT, 200U/ μ l M-MLV RT, template ribonucleic acid.42 DEG C of incubation 1h, 72 DEG C of 10min, of short duration centrifugation.
The specific implementation method of step (3) is：Using 25 μ l reaction systems, each sample arranges 3 parallel pipes, Suo Youkuo Increase reaction in triplicate above to ensure the reliability of result.Prepare following reaction system：SYBR Green polymerase chains 12.5 μ l of reaction system, 1 μ l of forward primer (5 μM/μ l), 1 μ l of reverse primer (5 μM/μ l), template cDNA2.0 μ l, without enzyme water 8.5μl；The forward primer sequence of amplification PPT1 genes is 5 '-ATGGTGTCAGATGAATATG-3 ', reverse primer sequences are 5 '- CATGTACTTATTGGCACAA-3’；Amplification GAPDH genes forward primer sequence be 5 '- TTTAACTCTGGTAAAGTGGATAT-3 ', reverse primer sequences are 5 '-GGTGGAATCATATTGGAACA-3 ', operations Carry out on ice.Amplification program is：95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 60s) * 45 circulations.Using SYBR Green as Fluorescent marker, in the enterprising performing PCR reaction of Light Cycler quantitative real time PCR Instruments, is analyzed by melt curve analysis and electrophoresis is true Determine purpose band, Δ Δ CT methods carry out relative quantification.
The advantages of the present invention are：(1) present invention firstly discloses PPT1 genes are related to osteosarcoma, PPT1 genes are expected to become the osteosarcomatous molecular marker of diagnosis, and provide new thinking to study osteosarcomatous molecule mechanism. (2) using detection gene expression mode diagnose osteosarcomatous presence or absence method it is sensitiveer, be conducive to disease early stage Diagnosis.
Description of the drawings：
Fig. 1 represent fluorescence real-time quantitative PCR determine in normal structure and osteosarcoma tissue PPT1 gene mRNAs it is relative Expression.
Specific embodiment：
The present invention is further illustrated with reference to specific embodiment, embodiments of the invention are only used for explaining the present invention, It is not intended to limit protection scope of the present invention.
Experimental technique used in following embodiments if no special instructions, is conventional method.
In following embodiments, material used, reagent etc., if no special instructions, commercially obtain.
Embodiment 1 screens the gene related to osteosarcoma
1.1NCBI GEO (Gene Expression Omnibus) data retrieval
GEO (Gene Expression Omnibus) data base is by NCBI (US National Biotechnology Information center) Exploitation safeguards, data base of the GEO data bases as maximum gene expression data, the data base based on chip data, in addition Also (ribosome sequence label connects for data such as SAGE (serial analysis of gene expression) data comprising some non-chip types, SARST Continuous analysis) data, MS (mass spectrum) data, proteome data and high-flux sequence data of new generation (MPSS, extensive parallel survey Sequence technology) etc..
A. search key：
(″osteosarcoma"[MeSH Terms]OR osteosarcoma[All Fields])AND″Homo sapi ens"[porgn]
B. the screening sample strategy in studying：
Limit research type to receive for the data set that " expression profiling by array " meets following standard Enter in our research：1. selected data collection must be the expression mRNA chip datas of full-length genome；2. these data come from The cell (without medicine irritation or transfected) of the biopsy or culture of osteosarcoma case group and matched group；3. this research considers Normalized or raw data set；4. selected data collection must be included more than more than 3 samples.Finally, there are 8 sets of chip datas Collection includes (shown in table 1) in our research.
The basic condition of 18 sets of osteosarcoma full-length genome data sets of table
1.2 osteosarcoma microarray data Meta analysis results
Initial data is carried out after background correction and standardization by DNA chip analysis software, using microarray significance Component software (significance analysis ofmicroarray, SAM) carries out the sieve of difference expression gene to 8 sets of data Choosing, it is 1.2 that multiple change (fold change, FC) is set during analysis, false positive rate (false discovery rate, FDR it is)≤0.05, filters out 3994 altogether, the gene 17 27 that wherein expression is raised, the gene that expression is lowered 3606.
2 QPCR of embodiment verifies candidate gene and osteosarcomatous relation
Based on the result analyzed to osteosarcoma microarray data Meta, according to the size of Pvalue, we select PPT1 Gene (Meta is analyzed and shown compared with normal structure, PPT1 genes up-regulated in osteosarcoma tissue) is verified.Collect 6 Individual osteosarcoma tissue sample, while collecting 6 normal structure samples, carries out the molecule life of classics using fluorescence real-time quantitative PCR Thing experimental verification (qRT-PCR), concrete operation step is as follows：
(1) RNA is extracted
After collecting sample, frozen tissue being put in the mortar of pre-cooling after liquid nitrogen, taking-up is ground, sample to be organized After this is powdered：
1. Trizol, room temperature preservation 5min are added；
2. the imitative 0.2ml of chlorination, uses forced oscillation centrifuge tube, fully mixes, places 5min-10min under room temperature；
3. draw upper strata aqueous phase (inhaling 70%) in another new centrifuge tube, to note not after 12000rpm high speed centrifugations 15min The protein substance between two-layer water phase is drawn onto.New pipe is moved into, isopyknic -20 DEG C of pre- cold isopropanols are added, it is fully reverse mixed It is even, it is placed in 10min on ice；
4. 12000rpm carefully discards supernatant at a high speed after 15min, adds 75% in the ratio of 1ml/ml Trizol DEPC washing with alcohol precipitation (4 DEG C of preservations), washing precipitate, vibration mixing, 12000rpm high speed centrifugations 5min at 4 DEG C；
5. ethanol liquid is discarded, places 5min fully to dry precipitation under room temperature, add the water dissolution that DEPC was processed to sink Form sediment；
6. RNA purity and concentration are measured with Nanodrop2000 ultraviolet spectrophotometers, it is frozen in -70 DEG C.
(2) reverse transcription
Reverse transcription synthesis cDNA is carried out with RT Buffer to 1 μ g total serum IgEs.Using 25 μ l reaction systems, each sample 1 μ g total serum IgEs are taken as template ribonucleic acid, following components is separately added in PCR pipe：DEPC water, 5 × RT Buffer, 10mmol/L dNTP, 0.1mmol/1DTT, 30 μm of mol/1Oligo dT, 200U/ μ l M-MLV, template ribonucleic acid.42 DEG C of incubations 1h, 72 DEG C of 10min, of short duration centrifugation.
(3) QPCR amplifications inspection
Using 25 μ l reaction systems, each sample arranges 3 parallel pipes, all amplified reactions in triplicate more than protecting The reliability of card result.Prepare following reaction system：12.5 μ l of SYBR Green polymerase chain reactions system, forward primer (5 μM/μ l) 1 μ l, reverse primer (5 μM/μ l) 1 μ l, template cDNA2.0 μ l, without 8.5 μ l of enzyme water；The forward direction of amplification PPT1 genes is drawn Thing sequence is 5 '-ATGGTGTCAGATGAATATG-3 ', and reverse primer sequences are 5 '-CATGTACTTATTGGCACAA-3 '；, expand The forward primer sequence for increasing GAPDH genes is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ', reverse primer sequences are 5 '- GGTGGAATCATATTGGAACA-3 ', operations are carried out on ice.Amplification program is：95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C 60s) * 45 circulations.Using SYBR Green as fluorescent marker, on Light Cycler fluorescence real-time quantitative PCR instrument Enter performing PCR reaction, purpose band is determined by melt curve analysis analysis and electrophoresis, Δ Δ CT methods carry out relative quantification, as a result such as Fig. 1 Shown, compared with normal structure, up-regulated of the PPT1 genes in osteosarcoma tissue is consistent with Meta analysis results.
The preparation of 3 osteosarcoma diagnostic kit of embodiment
According to PPT1 genes and osteosarcomatous dependency, kindred can be diagnosed by the expression of detection PPT1 genes Whether tumor occurs, and accordingly the invention provides one kind diagnoses osteosarcomatous test kit based on detection PPT1 gene expressions, this is examined Component in disconnected test kit is as follows：SYBR Green polymerase chain reactions system；Amplification PPT1 genes and GAPDH genes draw Thing pair.The forward primer sequence of amplification PPT1 genes is 5 '-ATGGTGTCAGATGAATATG-3 ', reverse primer sequences are 5 '- CATGTACTTATTGGCACAA-3’；The forward primer sequence of amplification GAPDH is 5 '-TTTAACTCTGGTAAAGTGGATAT- 3 ', reverse primer sequences are 5 '-GGTGGAATCATATTGGAACA-3 '.SYBR Green polymerase chain reactions system is included PCR buffer, dNTPs, SYBR Green fluorescent dyes.PCR buffer components are：25mM KCL, 2.5mM MgCL2、200mM (NH4)2SO4。
The preparation of 4 osteosarcoma diagnostic kit of embodiment
According to PPT1 genes and osteosarcomatous dependency, kindred can be diagnosed by the expression of detection PPT1 genes Whether tumor occurs, and accordingly the invention provides one kind diagnoses osteosarcomatous test kit based on detection PPT1 gene expressions, this is examined Component in disconnected test kit is as follows：SYBR Green polymerase chain reactions systems, amplification PPT1 genes and GAPDH genes draw Thing is to, M-MLV reverse transcription systems.The forward primer sequence of amplification PPT1 genes is 5 '-ATGGTGTCAGATGAATATG-3 ', instead It is 5 '-CATGTACTTATTGGCACAA-3 ' to primer sequence；Amplification GAPDH forward primer sequence be 5 '- TTTAACTCTGGTAAAGTGGATAT-3 ', reverse primer sequences are 5 '-GGTGGAATCATATTGGAACA-3 '.SYBR Green polymerase chain reactions system includes PCR buffer, dNTPs, SYBR Green fluorescent dyes.PCR buffer compositions For：25mM KCL、2.5mMMgCL2、200mM(NH4)2SO4.M-MLV reverse transcription system components are：T repeats oligonucleotide Oligo (dT), reverse transcription reaction liquid, M-MLV reverse transcriptases, RNase inhibitor, dNTPs.Reverse transcription reaction liquid component is：250mM Tris-HCL(PH8.3)、375mM KCL、15mM MgCL2, 50mM DTT.RNase inhibitor is non-for escherichia coli expression The recombinant protease of competitive inhibition RNase.
The preparation of 5 osteosarcoma diagnostic kit of embodiment
According to PPT1 genes and osteosarcomatous dependency, kindred can be diagnosed by the expression of detection PPT1 genes Whether tumor occurs, accordingly the invention provides one kind diagnoses osteosarcomatous test kit, reagent based on detection PPT1 gene expressions Component in box is as follows：SYBR Green polymerase chain reactions system, amplification PPT1 genes and GAPDH genes primer pair, RNA extracts reagents.Amplification PPT1 genes forward primer sequence be 5 '-ATGGTGTCAGATGAATATG-3 ', reverse primer sequence It is classified as 5 '-CATGTACTTATTGGCACAA-3 '；Amplification GAPDH forward primer sequence be 5 '- TTTAACTCTGGTAAAGTGGATAT-3 ', reverse primer sequences are 5 '-GGTGGAATCATATTGGAACA-3 '.SYBR Green polymerase chain reactions system includes PCR buffer, dNTPs, SYBR Green fluorescent dyes.PCR buffer compositions For：25mM KCL、2.5mM MgCL2、200mM(NH4)2SO4.RNA extracts reagents comprising Trizol, chloroform, isopropanol, 75% Ethanol.
The preparation of 6 osteosarcoma diagnostic kit of embodiment
According to PPT1 genes and osteosarcomatous dependency, kindred can be diagnosed by the expression of detection PPT1 genes Whether tumor occurs, accordingly the invention provides one kind diagnoses osteosarcomatous test kit, reagent based on detection PPT1 gene expressions Box component is as follows：Primer pair, the M-MLV of SYBR Green polymerase chain reactions system, amplification PPT1 genes and GAPDH genes Reverse transcription system, RNA extracts reagents.The forward primer sequence of amplification PPT1 genes is 5 '-ATGGTGTCAGATGAATATG-3 ', Reverse primer sequences are 5 '-CATGTACTTATTGGCACAA-3 '；Amplification GAPDH forward primer sequence be 5 '- TTTAACTCTGGTAAAGTGGATAT-3 ', reverse primer sequences are 5 '-GGTGGAATCATATTGGAACA-3 '.SYBR Green polymerase chain reactions system includes PCR buffer, dNTPs, SYBR Green fluorescent dyes.PCR buffer compositions For：25mM KCL、2.5mM MgCL2、200mM(NH4)2SO4.M-MLV reverse transcription system components are：T repeats oligonucleotide Oligo (dT), reverse transcription reaction liquid, M-MLV reverse transcriptases, RNase inhibitor, dNTPs.Reverse transcription reaction liquid component is： The Tris-HCL (PH8.3) of 250mM, the MgCL of KCL, 15mM of 375mM2, the DTT of 50mM.RNase inhibitor is escherichia coli The recombinant protease of the Noncompetition inhibition RNase of expression.RNA extracts reagents include Trizol, chloroform, isopropanol, 75% second Alcohol.

Claims (5)

1. the PPT1 genes of people prepare osteosarcoma diagnostic kit in purposes, it is characterised in that the diagnostic kit bag The polymerase chain reactions of Green containing SYBR system, the primer pair for expanding PPT1 genes and house-keeping gene；The SYBR Green polymerase chain reactions system is included：PCR buffer, dNTPs, SYBR Green fluorescent dyes.
2. purposes according to claim 1, it is characterised in that the sequence of the primer pair of the amplification PPT1 genes is as follows： Forward primer sequence is 5 '-ATGGTGTCAGATGAATATG-3 ', and reverse primer sequences are 5 '-CATGTACTTATTGGCACAA- 3’；The house-keeping gene is GAPDH, expand GAPDH genes forward primer sequence be 5 '- TTTAACTCTGGTAAAGTGGATAT-3 ', reverse primer sequences are 5 '-GGTGGAATCATATTGGAACA-3 '.
3. purposes according to claim 1, it is characterised in that the PCR buffer is included：25mM KCl、2.5mM MgCl2、200mM(NH4)2SO4。
4. the purposes according to any one of claim 1-3, it is characterised in that the diagnostic kit also includes M-MLV Reverse transcription system, the reverse transcription system are included：T repeats oligonucleotide Oligo dT, reverse transcription reaction liquid, M-MLV reverse transcriptions Enzyme, RNase inhibitor, dNTPs.
5. purposes according to claim 4, it is characterised in that the reverse transcription reaction liquid is included：250mM PH8.3's The MgCl of KCl, 15mM of Tris-HCl, 375mM2, 50mM DTT.
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Detection of Chromosomal Imbalances in Retinoblastoma by Matrix-Based Comparative Genomic Hybridization;Boris Zielinski et al;《GENES，CHROMOSOMES & CANCER》;20051231;第43卷;全文 *
The role of Serine/threonine protein phosphatase type 5 in the regulation of stress-induced signaling networks and cancer;Teresa Golden et al;《Cancer Metastasis Rev》;20081231;第27卷;第169-178页 *

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