CN105296658A - Application of ALKBH2 gene to diagnosis of cerebral ischemic stroke - Google Patents
Application of ALKBH2 gene to diagnosis of cerebral ischemic stroke Download PDFInfo
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Abstract
The invention discloses application of ALKBH2 gene to diagnosis of cerebral ischemic stroke. The ALKBH2 gene has low expression in cerebral ischemic stroke, and whether a patient has cerebral ischemic stroke or has a possibility of suffering from cerebral ischemic stroke or not can be judged by detecting the expression level of the ALKBH2 gene. According to the application of ALKBH2 gene to diagnosis of cerebral ischemic stroke, provided by the invention, the diagnostic sensitivity and specificity of cerebral ischemic stroke can be greatly improved, and an early diagnosis means of cerebral ischemic stroke is further provided.
Description
Technical field
The present invention relates to biological technical field, relate to the purposes of ALKBH2 gene in cerebral infarction diagnosis particularly.
Background technology
Cerebral infarction refers to the focal neurologic impairment syndrome that the brain local blood circulation obstacle occurred suddenly causes brain tissue ischemia anoxic to cause.There is the cerebral ischemia of Four types: transient ischemic attack, reversibility neurological dysfunction, advancing stroke, complete apoplexy.Cerebral infarction is one of three large dead diseases in world wide, is also disable reason in grownup first place.Because the pathogenesis of current cerebral infarction is not yet completely clear, and the means being used for the treatment of cerebral infarction are clinically limited, and not only sickness rate is high to make this disease, and lethality rate, disability rate and recurrence rate are also higher.
Cerebral infarction is the coefficient Complex Diseases of a kind of h and E factor, and along with the development with molecular genetics that completes of the Human Genome Project, the research of gene is in widespread attention, and the gene marker finding disease becomes the focus of current research.In this year, more and more research is thought, gene plays important regulating and controlling effect in the generation evolution of cerebral infarction, and therefore the relation of gene and cerebral infarction onset risk also receives much concern.
Comprise for the method for cerebral infarction diagnosis clinically at present: head CT and MRI scanning, cerebrovascular inspection (digital subtraction angiography DSA, CT or MR blood vessel imaging), TCD,transcranial Doppler.But the use of aforesaid method can not provide a kind of special and responsive diagnosis, although report now the gene that some are relevant to cerebral infarction, but be not applied to clinically, therefore find that a species specific cerebral infarction marker is applied to clinical diagnosis and has great importance.
Summary of the invention
In order to make up the deficiencies in the prior art, the object of the present invention is to provide a kind of molecular marker-ALKBH2 gene that can be used for cerebral infarction early diagnosis.Compare the diagnostic method of traditional cerebral infarction, use gene marker to carry out diagnosing ischemia cerebral apoplexy and there is promptness, specificity and susceptibility, make patient just can know risk in early days in disease, for risk height, take corresponding prevention and therapy measure.
To achieve these goals, the present invention adopts following technical scheme:
The invention provides a kind of ALKBH2 gene and the application of expression product in the product preparing diagnosing ischemia cerebral apoplexy thereof.
Further, the diagnostic products mentioned above comprises: by the expression level of RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip detection ALKBH2 gene and expression product thereof with the product of diagnosing ischemia cerebral apoplexy.
Further, the product of described RT-PCR diagnosing ischemia cerebral apoplexy at least comprises the primer of a pair specific amplified ALKBH2 gene; The product of described real-time quantitative PCR diagnosing ischemia cerebral apoplexy at least comprises the primer of a pair specific amplified ALKBH2 gene; The product of described immunodetection diagnosing ischemia cerebral apoplexy comprises: the antibody be combined with ALKBH2 protein-specific; The product of described in situ hybridization diagnosing ischemia cerebral apoplexy comprises: with the probe of the nucleic acid array hybridizing of ALKBH2 gene; The product of described chip diagnosing ischemia cerebral apoplexy comprises: protein chip and gene chip; Wherein, protein chip comprises the antibody be combined with ALKBH2 protein-specific, and gene chip comprises the probe with the nucleic acid array hybridizing of ALKBH2 gene.
Further, described diagnostic products comprises chip and/or test kit; Wherein said chip comprises gene chip, protein chip; Described test kit comprises gene detecting kit and protein immunization detection kit.Described gene detecting kit comprises SYBRGreen polymerase chain reaction system, primer pair for increase ALKBH2 gene and house-keeping gene; Described SYBRGreen polymerase chain reaction system comprises: PCR damping fluid, dNTPs, SYBRGreen fluorescence dye.
The invention provides a kind of product of diagnosing ischemia cerebral apoplexy, described product can carry out diagnosing ischemia cerebral apoplexy by the expression level detecting ALKBH2 gene.
Further, product recited above comprises chip or test kit.Wherein, described chip comprises gene chip, protein chip; Described gene chip comprises solid phase carrier and is fixed on the oligonucleotide probe on solid phase carrier, and described oligonucleotide probe comprises the oligonucleotide probe for ALKBH2 gene for detecting ALKBH2 gene transcription level; Described protein chip comprises solid phase carrier and is fixed on the specific antibody of the ALKBH2 albumen on solid phase carrier; Described test kit comprises gene detecting kit and protein immunization detection kit; Described gene detecting kit comprises the reagent for detecting ALKBH2 gene transcription level; Described protein immunization detection kit comprises the specific antibody of ALKBH2 albumen.Wherein, described gene chip can be used for detecting the expression level of the multiple genes (such as, relevant to cerebral infarction multiple genes) comprising ALKBH2 gene.Described protein chip can be used for detecting the expression level of the multiple protein (such as relevant to cerebral infarction multiple protein) comprising ALKBH2 albumen.By being detected by multiple mark with cerebral infarction simultaneously, the accuracy rate of cerebral infarction diagnosis greatly can be improved.
Further, above, described reagent comprises the reagent used needed in RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip method detection ALKBH2 gene expression dose process.Preferably, described reagent comprises primer for ALKBH2 gene and/or probe.
Further, described gene detecting kit comprises SYBRGreen polymerase chain reaction system, primer pair for increase ALKBH2 gene and house-keeping gene; Described SYBRGreen polymerase chain reaction system comprises: PCR damping fluid, dNTPs, SYBRGreen fluorescence dye.The primer pair sequence of described amplification ALKBH2 gene is as follows: forward primer sequence is 5 '-TGGACAGACCTTCAACTT-3 '; Reverse primer sequences is 5 '-TTCATCATCTCGGTGCTC-3 '; Described house-keeping gene is GAPDH, and the primer sequence of amplification GAPDH is to as follows: forward primer sequence is 5 '-CTCTGGTAAAGTGGATATTGT-3 '; Reverse primer sequences is 5 '-GGTGGAATCATATTGGAACA-3 '.
Further, described gene detecting kit also comprises M-MLV reverse transcription system, and described reverse transcription system comprises T and repeats oligonucleotide OligodT, reverse transcription reaction liquid, M-MLV reversed transcriptive enzyme, RNA enzyme inhibitors, dNTPs.Described gene detecting kit can be used for detecting the expression level of the multiple genes (such as, relevant to cerebral infarction multiple genes) comprising ALKBH2 gene.
Can be DNA, RNA, DNA-RNA mosaic, PNA or other derivative with the probe of the nucleic acid array hybridizing of ALKBH2 gene in the present invention.The length of described probe does not limit, if complete specific hybrid, with object nucleotide sequence specific binding, any length can.The length of described probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the length of described probe can grow to 60,80,100,150,300 base pairs or longer, even whole gene.Because different probe length has different impacts to hybridization efficiency, signal specificity, the length of described probe is at least 14 base pairs usually, the longlyest generally be no more than 30 base pairs, best with 15-25 base pair with the length of object nucleotide sequence complementary.Described probe self-complementary sequences most preferably less than 4 base pairs, in order to avoid affect hybridization efficiency.
The specific antibody of the albumen of ALKBH2 described in the present invention comprises monoclonal antibody, polyclonal antibody.The specific antibody of described ALKBH2 albumen comprises complete antibody molecule, any fragment of antibody or modification, such as, and chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc.As long as described fragment can retain the binding ability with ALKBH2 albumen.Be well known to a person skilled in the art for detecting the preparation of the antibody of protein level, and the present invention can use any method to prepare described antibody, as described in fragment or recombinant DNA technology can be utilized to synthesize by chemical method de novo synthesis.
In the context of the present invention, " ALKBH2 gene " comprises the polynucleotide of any function equivalent of people ALKBH2 gene and people ALKBH2 gene.ALKBH2 gene comprises and has more than 70% homology with ALKBH2 gene (NC_000012.12) DNA sequence dna in current international common core sequence databank GeneBank, and coding identical function protein DNA sequence;
Preferably, the encoding sequence of ALKBH2 gene comprises any one DNA molecular following:
(1) DNA sequence dna shown in SEQ ID NO.1;
(2) DNA sequence dna limited with (1) is under strict conditions hybridized and identical function protein DNA sequence of encoding;
(3) DNA sequence dna limited with (1) or (2) has 70%, preferably, more than 90% homology, and coding identical function protein DNA molecule.
In specific embodiment of the invention scheme, the encoding sequence of described ALKBH2 gene is the DNA sequence dna shown in SEQIDNO.1.
ALKBH2 gene of the present invention can be natural or synthetic, or uses the vector-transfected cell can expressing the DNA fragmentation of ALKBH2 to obtain.Described in described carrier, carrier comprises virus vector, carrier for expression of eukaryon.
Virus vector can be any suitable carrier, includes but not limited to retroviral vector, adenovirus carrier, adeno-associated virus (AAV) carrier, simplexvirus (such as hsv, vaccinia virus and Epstein-Barr virus) carrier, alphavirus vectors.
Carrier for expression of eukaryon can be any suitable expression vector, include but not limited to pCMV-Myc expression vector, pcDNA3.0 expression vector, pcDNA3.1 expression vector, pEGFP expression vector, pEFBos expression vector, pTet expression vector, pTRE expression vector or the carrier through transforming on the basis of known expression vector, such as pBin438, pCAMBIA1301 etc.
In the context of the present invention, ALKBH2 gene expression product comprises the partial peptide of people ALKBH2 albumen and people ALKBH2 albumen.The partial peptide of described ALKBH2 albumen contains the functional domain relevant to cerebral infarction.
" ALKBH2 albumen " comprises any function equivalent of ALKBH2 albumen and ALKBH2 albumen.Described function equivalent comprises ALKBH2 albumen conservative variation's protein or its active fragments, or its reactive derivative, allelic variant, natural mutation, induced mutants, can with the protein coded by the DNA of the DNA hybridization of people ALKBH2 under high or low stringent condition.
Preferably, ALKBH2 albumen is the protein with following amino acid sequences:
(1) protein be made up of the aminoacid sequence shown in SEQ ID NO.2;
(2) aminoacid sequence shown in SEQIDNO.2 had the protein derivative by the aminoacid sequence shown in SEQIDNO.2 of identical function with the aminoacid sequence shown in SEQIDNO.2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation.The amino acid whose number replacing, lack or add is generally 1-50, preferably 1-30, and more preferably 1-20,1-10 is individual best.
(3) with the aminoacid sequence shown in SEQIDNO.2, there is at least 80% homology (being also called sequence iden), preferably, with the aminoacid sequence shown in SEQIDNO.2 at least about 90% to 95% homology, be often 96%, 97%, 98%, 99% homology aminoacid sequence form polypeptide.
In specific embodiment of the invention scheme, described ALKBH2 albumen is the protein with the aminoacid sequence shown in SEQIDNO.2.
Usually, it is known that in a protein one or more amino acid whose modification can not affect the function of protein.Those skilled in the art can approve the amino acid that changes single amino acids or little per-cent or be conservative modifications to indivedual interpolations of aminoacid sequence, disappearance, insertion, replacement, and wherein the change of protein produces the protein with identity function.Intimate amino acid whose Conservative substitution tables is provided to be well known in the art.
By adding the fusion rotein that the example of the protein of an amino acid or multiple Modification of amino acid residues is ALKBH2 albumen.Peptide or protein with ALKBH2 protein fusion are not limited, as long as the fusion rotein of gained retains the biologic activity of ALKBH2 albumen.
ALKBH2 albumen of the present invention also comprises the non-conservative modification to the aminoacid sequence shown in SEQIDNO.2, as long as the protein through modifying still can retain the biologic activity of ALKBH2 albumen.The amino acid number suddenlyd change in this type of modifying protein normally 10 or less, such as 6 or less, such as 3 or less.
In the context of the present invention, " diagnosing ischemia cerebral apoplexy " had both comprised and had judged whether experimenter has suffered from cerebral infarction, also comprised and judge whether experimenter exists the risk suffering from cerebral infarction.
In the context of the present invention, " treatment cerebral infarction " comprises generation and the recurrence of any symptom eliminating, alleviate, alleviate, reverse or prevent or postpone illness, namely comprises the therapeutic intervention to disease and Primary preventive intervention.
Advantage of the present invention and beneficial effect:
Late Cambrian of the present invention ALKBH2 genetic expression is relevant to cerebral infarction, by detecting the expression of ALKBH2 in experimenter's blood, can judge whether experimenter suffers from cerebral infarction or judge whether experimenter exists the risk suffering from cerebral infarction, thus instruct clinicist to provide prevention scheme or treatment plan to experimenter.
Present invention finds a kind of new molecular marked compound-ALKBH2 gene of cerebral infarction, compare traditional detection means, gene diagnosis more in time, more special, sensitiveer, the early diagnosis of cerebral infarction can be realized, thus reduce the mortality ratio of cerebral infarction.
Accompanying drawing explanation
Fig. 1 display utilizes QPCR to detect the expression of ALKBH2 gene in ischemic cerebral stroke patients;
Concrete embodiment
Below in conjunction with drawings and Examples, the present invention is further detailed explanation.Following examples are only not used in for illustration of the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in embodiment, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.
Embodiment 1 screens the gene marker relevant to cerebral infarction
1, sample collection
The routine normal human blood of each collection 10 and ischemic cerebral stroke patients blood sample, obtaining all by the agreement of Ethics Committee of above-mentioned all samples.
2, the preparation of RNA sample and mass analysis
The preparation of 2.1RNA sample
Use the RNA of Promega company to extract test kit and extract total serum IgE.Concrete steps are as follows:
1) get 1ml and be collected in whole blood in the test tube of heparin or EDTA process, put in sterile centrifugation tube;
2) collect hemocyte: 3000rpm (400g) centrifugal 5min, siphon away supernatant from sample top carefully;
3) add 1ml hemocyte lysate, careful suction is put 4-5 time, resuspended throw out;
4) the centrifugal 5min of 3000rpm;
5) repeating step 3), 4) twice (totally three times);
6) avoid cell precipitation thing, carefully siphon away nearly all supernatant, only retain 100 μ l supernatant liquors; Check that BME has been added in RNA lysate, then add 175 μ lRNA lysates in cell, inhale and put resuspended and lysing cell;
7) add 350 μ lRNA diluents, put upside down mixing 3-4 time;
8) 70 DEG C of water-bath 3min are placed in;
9) the centrifugal 10min of 13000g under room temperature;
10) limpid lysate is transferred in a sterile centrifugation tube;
11) in cleared lysate, add 200 μ l95% ethanol, inhale with liquid-transfering gun and put 3-4 time with mixing; This mixture is transferred in centrifugal column assembly, the centrifugal 1min of 13000g;
12) centrifugal column is taken down from centrifugal column assembly, discard the liquid in collection tube, centrifugal column is installed onto on collection tube, check that RNAWashSolution uses alcohol dilution, add 600 μ lRNA washingss in centrifugal column assembly, the centrifugal 1min of 13000g;
13) discard the liquid in collection tube, centrifugal column is installed onto on collection tube, the DNase mixtures incubated of the fresh preparation of 50 μ l is directly added on the film in centrifugal column;
14) incubated at room temperature 15min, adds 200 μ lDNA enzyme stop buffers (confirming to add ethanol), the centrifugal 1min of 13000g in centrifugal column;
15) 600 μ lRNA washingss (adding ethanol) are added, 13000g, centrifugal 1min;
16) empty collection tube, in centrifugal column, add 250 μ lRNA washingss (adding ethanol), high speed centrifugation 2min;
17) centrifugal column is transferred on wash-out pipe from collection tube, on film, add 100 μ l nuclease free water, put centrifugal column assembly into whizzer and make the lid facing outwards of wash-out pipe, the centrifugal 1min of 13000g, abandon centrifugal column, build the wash-out pipe filling RNA, be stored in-70 DEG C.
The mass analysis (NanoDrop1000 spectrophotometer) of 2.2RNA sample
NanoDrop1000 spectrophotometer detects RNA sample, the sample requirement of RNA-seq order-checking: OD260/OD280 is 1.8-2.2.
The mass analysis (AgilentTechnologies2100Bioanalyzer) of 2.3RNA sample
The RNA of said extracted is carried out agarose gel electrophoresis, AgilentTechnologies2100Bioanalyzer detects RNA sample quality, observation 28SrRNA and 18SrRNA master tape obviously, nothing is degraded, RNA Perfection Index is qualified, concentration meets the requirements of the requirement meeting RNA-seq order-checking cDNA library structure, may be used for library construction and order-checking.
3, high-throughput transcript profile order-checking
The 3.1RNA-seq section of reading is located
First the low-quality section of reading is removed and obtain the clean section of reading, then utilize TopHatv1.3.1 will clean fragment to mate with reference to genome (hg19) with UCSCH.sapiens, the index built in advance of H.sapiensUCSChg19 version is downloaded from TopHat homepage, and as reference genome, when utilizing TopHat to mate with genome, each section of reading (defaulting to 20) is allowed to have multiple coupling site, maximum 2 mispairing.TopHat sets up possible shearing site storehouse according to exon region and GT-AG shear signal, will not navigate to the genomic section of reading navigate on genome according to these shearing site storehouses.We use the system default parameter of TopHat method.
3.2 transcript abundance assessments
What match reads segment file by Cufflinksv1.0.3 process, and RNA-seq segment number is carried out the relative abundance of standardized calculation transcript by Cufflinksv1.0.3.FPKM value refers in each 1,000,000 sequenced fragments the segment number matching the long exon region of specific gene 1kb.The fiducial interval of FPKM estimated value is calculated by Bayesian inference method.The GTF comment file of the reference that Cufflinks uses downloads (Homo_sapiens.GRCh37.63.gtf) from Ensembl database.
The detection of 3.3 difference expression genes
By the EnsemblGTF file of download be transferred to Cuffdiff by the source document that TopHat mates, Cuffdiff uses original matching files again to estimate the gene expression abundance of the transcript listed in GTF file, and checkout discrepancy is expressed.In Cuffidff exports, only have q value < 0.01, test display is successfully more just considered to differential expression.
4, result
RNA-seq result shows, and the expression amount of ALKBH2 gene in ischemic cerebral stroke patients blood is significantly lower than the expression amount of normal people.
The differential expression of embodiment 2QPCR sequence verification ALKBH2 gene
1, ALKBH2 gene is selected to carry out large sample QPCR checking according to the detected result of high-flux sequence.According to the sample collection way selection ischemic cerebral stroke patients blood in embodiment 1 and each 80 examples of normal human blood.
2, RNA extraction step is with embodiment 1.
3, reverse transcription: use the Reverse Transcription box of TAKARA company to operate.Concrete steps are as follows:
(1) get total serum IgE 2 μ g and carry out reverse transcription, add Oligo (dT) 2 μ l, fully mix; 70 DEG C of water-baths; Ice bath 2-3min immediately after 5min;
(2) build 25 μ l reaction systems, comprising 5 × RT Buffer 5 μ l, dNTP (2.5mM) 5 μ l, RNasin40U/ μ l, M-MLV200U/ μ l, mend nuclease free water to 25 μ l;
After (3) 42 DEG C of water-bath 60min, 95 DEG C of water-bath 5min are with deactivation M-MLV;
(4)-20 DEG C store for future use.
4, QPCR amplification
(1) design of primers
According to the encoding sequence design QPCR amplimer of ALKBH2 gene and GAPDH gene in Genebank, synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Concrete primer sequence is as follows:
ALKBH2 gene:
Forward primer is 5 '-TGGACAGACCTTCAACTT-3 ' (SEQIDNO.3);
Reverse primer is 5 '-TTCATCATCTCGGTGCTC-3 ' (SEQIDNO.4).
GAPDH gene:
Forward primer is 5 '-CTCTGGTAAAGTGGATATTGT-3 ' (SEQIDNO.5);
Reverse primer is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQIDNO.6).
(2) PCR reaction system is prepared according to table 1:
Wherein, SYBRGreen polymerase chain reaction system is purchased from Invitrogen company.
Table 1PCR reaction system
| Reagent | Volume |
| Forward primer | 1μl |
| Reverse primer | 1μl |
| SYBR Green polymerase chain reaction | 12.5μl |
| System template | 2μl |
| Deionized water | Supply 25 μ l |
(3) PCR reaction conditions: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 60s) × 45 circulations.Using SYBRGreen as fluorescent marker, in the enterprising performing PCR reaction of LightCycler quantitative real time PCR Instrument, by melt curve analysis analysis and electrophoresis determination object band, Δ Δ CT method carries out relative quantification.
5, statistical method
Experiment employing repeats experiment for 3 times, result data is all represent in the mode of mean+SD, use SPSS13.0 statistical software to carry out statistical study, difference between the two adopts t inspection, thinks to have statistical significance as P<0.05.
6, result
As shown in Figure 1, compared with normal human blood, the down-regulated expression of ALKBH2 gene in ischemic cerebral stroke patients blood, difference has statistical significance (P<0.05) to result, consistent with RNA-sep result.
The explanation of above-described embodiment is just for understanding method of the present invention and core concept thereof.It should be pointed out that for the person of ordinary skill of the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and also will fall in the protection domain of the claims in the present invention.
Claims (10)
1.ALKBH2 gene and the application of expression product in the product preparing diagnosing ischemia cerebral apoplexy thereof.
2. application according to claim 1, it is characterized in that, described product comprises: by the expression level of RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip detection ALKBH2 gene and expression product thereof with the product of diagnosing ischemia cerebral apoplexy.
3. application according to claim 2, is characterized in that, the product of described RT-PCR diagnosing ischemia cerebral apoplexy at least comprises the primer of a pair specific amplified ALKBH2 gene; The product of described real-time quantitative PCR diagnosing ischemia cerebral apoplexy at least comprises the primer of a pair specific amplified ALKBH2 gene; The product of described immunodetection diagnosing ischemia cerebral apoplexy comprises: the antibody be combined with ALKBH2 protein-specific; The product of described in situ hybridization diagnosing ischemia cerebral apoplexy comprises: with the probe of the nucleic acid array hybridizing of ALKBH2 gene; The product of described chip diagnosing ischemia cerebral apoplexy comprises: protein chip and gene chip; Wherein, protein chip comprises the antibody be combined with ALKBH2 protein-specific, and gene chip comprises the probe with the nucleic acid array hybridizing of ALKBH2 gene.
4. the application according to any one of claim 1-3, is characterized in that, described product comprises chip and/or test kit; Wherein said chip comprises gene chip, protein chip; Described test kit comprises gene detecting kit and protein immunization detection kit.
5. application according to claim 4, is characterized in that, described gene detecting kit comprises SYBRGreen polymerase chain reaction system, primer pair for increase ALKBH2 gene and house-keeping gene; Described SYBRGreen polymerase chain reaction system comprises: PCR damping fluid, dNTPs, SYBRGreen fluorescence dye.
6. a product for diagnosing ischemia cerebral apoplexy, is characterized in that, described product can carry out diagnosing ischemia cerebral apoplexy by the expression level detecting ALKBH2 gene.
7. product according to claim 6, is characterized in that, described product comprises chip or test kit; Wherein, described chip comprises gene chip, protein chip; Described test kit comprises gene detecting kit and protein immunization detection kit.
8. product according to claim 7, is characterized in that, described gene detecting kit comprises SYBRGreen polymerase chain reaction system, primer pair for increase ALKBH2 gene and house-keeping gene; Described SYBRGreen polymerase chain reaction system comprises: PCR damping fluid, dNTPs, SYBRGreen fluorescence dye.
9. product according to claim 8, is characterized in that, the primer pair sequence of described amplification ALKBH2 gene is as follows: forward primer sequence is 5 '-TGGACAGACCTTCAACTT-3 '; Reverse primer sequences is 5 '-TTCATCATCTCGGTGCTC-3 '; Described house-keeping gene is GAPDH, and the primer sequence of amplification GAPDH is to as follows: forward primer sequence is 5 '-CTCTGGTAAAGTGGATATTGT-3 '; Reverse primer sequences is 5 '-GGTGGAATCATATTGGAACA-3 '.
10. product according to claim 8 or claim 9, it is characterized in that, described gene detecting kit also comprises M-MLV reverse transcription system, and described reverse transcription system comprises T and repeats oligonucleotide OligodT, reverse transcription reaction liquid, M-MLV reversed transcriptive enzyme, RNA enzyme inhibitors, dNTPs.
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