CN103060351A - Gene marker for detecting cancer tissue and para-carcinoma tissue of primary liver cancer - Google Patents
Gene marker for detecting cancer tissue and para-carcinoma tissue of primary liver cancer Download PDFInfo
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Abstract
The invention relates to a gene marker for detecting a cancer tissue and a para-carcinoma tissue of primary liver cancer. The inventor firstly screens out 33 specific genes by virtue of a statistical method by analyzing a whole genome expression profile of the cancer tissue and the para-carcinoma tissue of a patient suffering from the primary liver cancer. The test proves that the specific markers can be very effectively used for distinguishing the cancer tissue and para-carcinoma tissue of the patient suffering from the primary liver cancer.
Description
Technical field
The invention belongs to the medicine and pharmacology field; More specifically, the present invention relates to for detection of the cancerous tissue of primary hepatocarcinoma and the gene marker of cancer beside organism.
Background technology
The genesis of anything all is under the internal and external reasons acting in conjunction, and is interior because main, outer because auxiliary.Gene is as the hereditary medium of life, organism sick, old,, be in the status of basic internal cause in dead.Overwhelming majority gene is by transcribing product nucleus ribosomal ribonucleic acid mRNA, and translation generates protein and brings into play biological function again.Although the function of gene mainly is by the embodying of protein, but still the content of transcription product mRNA that can be by gene and part reflection.
Full genomic expression spectrum chip contains more than 20,000 probes, utilize the content of principle contained various mRNA in full genomic level detection sample of dna double chain homologous complementary, therefore can be at one time the transcriptional level of the gene in the full genome range in the sample to be tested be detected, and then infer gene function.
Primary hepatocarcinoma is one of Chinese modal malignant tumour, and annual new cases account for the whole world 50%.The grade malignancy of liver cancer is high, and development is treated difficulty rapidly, and case fatality rate is high.Therefore, the as early as possible diagnosis of liver cancer just more seems urgent.
Yet, up to now, this area is for very few with the closely-related gene understanding of primary hepatocarcinoma, so this area is found generation, transfer, the recurrence for diagnosing liver cancer or detected relevant gene marker in the urgent need to separating further various and the closely-related gene of primary hepatocarcinoma.
Summary of the invention
The object of the present invention is to provide for detection of, distinguish the cancerous tissue of primary hepatocarcinoma and the gene marker of cancer beside organism.
In a first aspect of the present invention, a kind of polynucleotide collection is provided, described polynucleotide collection comprises the polynucleotide (preferably, the polynucleotide by the following albumen of coding form) of the following albumen of encoding:
Fiber gelatinized protein 3, CD209 molecule, estrogen receptor 1; the CD5 quasi-molecule, kynuric acid 3-hydroxylase, N-acyltransferase; carry ester gp F, iron is transferred plain antibacterial peptide, GYS2; steroid-5-alpha-reductase enzyme 2 is urged renal cortex element hormone in conjunction with albumen, ethanol dehydrogenase 4; Cytochrome P450 family 2 subfamily C polypeptide 19; Cytochrome P450 family 2 subfamily C polypeptide 8, coagulation factor 9, Cytochrome P450 family 2 subfamily C polypeptide 9; Metallothionein 1 G; phosphoenolpyruvate carboxykinase, the macrophage receptor of one-tenth collagen structure, Metallothionein 1 H; Afamin; glycine-N-acyltransferase, retinol dehydrogenase 16, GNMT; first ammonium second lactone-homocysteine methyl transferase; tyrosine aminotransferase, ApoA5, serine dehydratase; secretor type phosphorprotein 2; L-Ala-oxoethanoic acid transaminase 2 sample albumen 1, Cytochrome P450 family 26 subfamily A polypeptide 1,17-beta hydroxysteroid dehydrogenase 6 homologous proteins and minopontin.
In a preference; described polynucleotide are concentrated; the described fiber gelatinized protein 3 of encoding; the CD209 molecule; estrogen receptor 1; the CD5 quasi-molecule; kynuric acid 3-hydroxylase; the N-acyltransferase; carry ester gp F, iron is transferred plain antibacterial peptide, GYS2; steroid-5-alpha-reductase enzyme 2; urge renal cortex element hormone in conjunction with albumen, ethanol dehydrogenase 4, Cytochrome P450 family 2 subfamily C polypeptide 19; Cytochrome P450 family 2 subfamily C polypeptide 8; coagulation factor 9, Cytochrome P450 family 2 subfamily C polypeptide 9, Metallothionein 1 G; phosphoenolpyruvate carboxykinase; become the macrophage receptor of collagen structure, Metallothionein 1 H, Afamin; glycine-N-acyltransferase; retinol dehydrogenase 16, GNMT, first ammonium second lactone-homocysteine methyl transferase; tyrosine aminotransferase; ApoA5, serine dehydratase, secretor type phosphorprotein 2; L-Ala-oxoethanoic acid transaminase 2 sample albumen 1; Cytochrome P450 family 26 subfamily A polypeptide 1, the polynucleotide of 17-beta hydroxysteroid dehydrogenase 6 homologous proteins and minopontin are respectively: sequence such as GenBank accession number NM_003665.2, NM_021155.3; NM_021155.3; NM_005894.2, NM_003679.3, NM_000015.2; NM_001638.2; NM_021175.2, NM_021957.3, NM_000348.3; NM_001882.3; NM_000670.3, NM_000769.1, NM_000770.3; NM_000133.3; NM_000771.3, NM_005950.1, NM_002591.3; NM_006770.3; NM_005951.2, NM_001133.2, NM_201648.2; NM_003708.3; NM_018960.4, NM_001713.2, NM_000353.2; NM_052968.3; NM_006843.2, NM_006944.2, NM_031279.2; NM_000783.3, the polynucleotide shown in NM_003725.2 and the NM_001040058.1.
In another aspect of this invention, provide the purposes of described polynucleotide collection, for the preparation of measuring (or differentiation) cancerous tissue of primary hepatocarcinoma and the detection reagent of cancer beside organism or test kit.
In a preference, described reagent is the primer pair that the described polynucleotide of specific amplification are concentrated each polynucleotide; Or the described polynucleotide of specific recognition are concentrated the probe of each polynucleotide.
In another aspect of this invention, provide a kind of for measuring (or differentiation) cancerous tissue of primary hepatocarcinoma and reagent of cancer beside organism, it is specific recognition sequence such as GenBank accession number NM_003665.2, NM_021155.3, NM_021155.3, NM_005894.2, NM_003679.3, NM_000015.2, NM_001638.2, NM_021175.2, NM_021957.3, NM_000348.3, NM_001882.3, NM_000670.3, NM_000769.1, NM_000770.3, NM_000133.3, NM_000771.3, NM_005950.1, NM_002591.3, NM_006770.3, NM_005951.2, NM_001133.2, NM_201648.2, NM_003708.3, NM_018960.4, NM_001713.2, NM_000353.2, NM_052968.3, NM_006843.2, NM_006944.2, NM_031279.2, NM_000783.3, the probe of the polynucleotide shown in NM_003725.2 or the NM_001040058.1, the nucleotide sequence of described probe is shown in SEQ ID NO:1-33 is arbitrary.
In another aspect of this invention, provide a kind of gene chip, described gene chip comprises:
Solid phase carrier; And
Be fixed in order the probe on the described solid phase carrier, described probe specificity ground is corresponding to sequence such as GenBank accession number NM_003665.2, NM_021155.3, NM_021155.3, NM_005894.2, NM_003679.3, NM_000015.2, NM_001638.2, NM_021175.2, NM_021957.3, NM_000348.3, NM_001882.3, NM_000670.3, NM_000769.1, NM_000770.3, NM_000133.3, NM_000771.3, NM_005950.1, NM_002591.3, NM_006770.3, NM_005951.2, NM_001133.2, NM_201648.2, NM_003708.3, NM_018960.4, NM_001713.2, NM_000353.2, NM_052968.3, NM_006843.2, NM_006944.2, NM_031279.2, NM_000783.3, the polynucleotide shown in NM_003725.2 or the NM_001040058.1.
In a preference, the nucleotide sequence of described probe is shown in SEQ ID NO:1-33 is arbitrary.
In another preference, described probe contains: complementary land and/or the joining region that links to each other with solid phase carrier.
In another aspect of this invention, provide the purposes of described gene chip, for the preparation of measuring (or differentiation) cancerous tissue of primary hepatocarcinoma and test kit of cancer beside organism.
In another aspect of this invention, provide a kind of mensuration (or differentiation) cancerous tissue of primary hepatocarcinoma and the test kit of cancer beside organism, contain described gene chip in the described test kit.
In a preference, also comprise in the described test kit: nucleic acid extract, nitrite ion or hybridization solution.
Other side of the present invention is because the disclosure of this paper is apparent to those skilled in the art.
Description of drawings
Fig. 1, with the scatter diagram of sample (blue dot or dark point) in three-dimensional space by the cancer sample after the MDS algorithm conversion (red point or light point) and the cancer.Wherein, Cancer represents the cancer sample, and Pericancerous represents the other sample of cancer.
Embodiment
The inventor uses statistical method by the full genomic expression spectrum of cancerous tissue sample and cancer beside organism's sample in the same Patients with Primary of detection source, therefrom filters out first 33 specific genes.Through testing identity, these specific gene markers can be used for distinguishing primary hepatic carcinoma patient's cancerous tissue and cancer beside organism very effectively.
Gene marker and uses thereof
Primary hepatocarcinoma is one of modal malignant tumour of China.The inventor adopts the method for chip hybridization to obtain the full genomic expression spectrum of cancerous tissue sample and cancer beside organism's sample of primary hepatic carcinoma, by comparing the express spectra of two kinds of tissues, obtains the gene of differential expression between cancer and cancer beside organism.Pick out the particularly significant part of difference in these differential genes, using the screening of C-SVC sorter (gaussian kernel function) algorithm to obtain classify accuracy is a classifiers (containing 33 genes) of 95%.By the sorter of these 33 genomic constitutions, measurable sample is from cancerous tissue or cancer beside organism, and its prediction accuracy reaches 87%.Can become these 33 Data minings small-sized gene chip or RT-PCR test kit to be used for the differential diagnosis of liver cancer.
Described 33 genes are as follows: FCN3, CD209, ESR1, CD5L, KMO, NAT2, APOF, HAMP, GYS2, SRD5A2, CRHBP, ADH4, CYP2C19, CYP2C8, F9, CYP2C9, MT1G, PCK1, MARCO, MT1H, AFM, GLYAT, RODH-4, GNMT, BHMT, TAT, APOA5, SDS, SPP2, AGXT2L1, CYP26A1, RODH, SPP1.
Based on the inventor's new discovery, can be with above-mentioned 33 genes as measuring (or differentiation) cancerous tissue of primary hepatocarcinoma and mark of cancer beside organism (marker).By analyzing the expression of these genes, thereby learn the type of determining tissue to be measured, for diagnosis or the prognosis of disease provides foundation.
Can adopt various technology to detect the expression of said gene, these technology all comprise in the present invention.On the gene level, detection can be for cDNA, also can be for genomic dna, or for mRNA; Available prior art is such as (but being not limited to): biochip technology, probe hybridization technology, polymerase chain reaction (PCR), Southern blotting, dna sequence analysis etc.
As a kind of selection mode of the present invention, come expression and the expression amount of said gene in the analytic sample by quantitative or semiquantitative polymerase chain reaction (PCR) method, thereby can judge.Preferably, realize detecting by RT-PCR.
Gene chip
As a kind of optimal way of the present invention, adopt biochip technology to detect the expression of said gene.Comprise the probe for said gene on the described gene chip; Preferred, comprise the probe for two or more described gene on the described gene chip; Most preferred, comprise the probe for described 33 kinds of genes on the described gene chip.
Gene chip of the present invention comprises solid phase carrier and is fixed in order oligonucleotide probe on the described solid phase carrier that described oligonucleotide probe (is more particularly 20-80 by 10-100; Be more particularly 30-70) the continuous nucleotide composition.Described probe can also comprise the poly-poly-deoxythymidylic acid of one section amido modified 1-30 (poly-dT) at its 5 ' end.
Described solid phase carrier can adopt the various common used materials in gene chip field, such as but not limited to nylon membrane, and the slide of the slide of modifying through active group (such as aldehyde radical, amino, the fine acidic group of different sulphur etc.) or silicon chip, unmodified, plastic sheet etc.
The preparation of gene chip of the present invention can be adopted the conventional manufacture method of biochip.For example, if what solid phase carrier adopted is to modify slide or silicon chip, 5 ' end of probe contains amido modified poly-dT string, oligonucleotide probe can be mixed with solution, then point sample instrument is being modified slide or silicon chip with its point, be arranged in predetermined sequence or array, then spending the night by placement fixes, and just can obtain gene chip of the present invention.If it is amido modified that oligonucleotide probe does not contain, then its preparation method also can reference: Wang Shenwu chief editor's " gene diagnosis technology-on-radiation operational manual "; J.L.erisi, V.R.Iyer, P.O.BROWN.Exploring the metabolic and genetic control of gene expression on a genomic scale.Science.1997; 278:680 and Ma Li people, the Jiang Zhonghua chief editor. biochip. Beijing: Chemical Industry Press, 2000,1-130.
Another aspect, the present invention also provides a kind of method by gene expression profile described in the genechip detection people tissue, comprises step:
(1) provides the RNA sample that separates from people's tissue, at described RNA marker is set;
(2) RNA with (1) contacts with described chip, makes the oligonucleotide probe generation hybridization on described RNA and the solid phase carrier, thereby forms " oligonucleotide probe-RNA " binary complex at solid phase carrier;
(3) detect the marker of the binary complex of (2) formation, thereby determine the express spectra of corresponding gene in people's tissue.
Solid-phase hybridization between RNA of the present invention and the chip carries out according to the classical way of this area, and the general personnel in this area easily determine relevant damping fluid, probe and the optimum condition of concentration of specimens, prehybridization temperature, hybridization temperature and time etc. according to experience.Perhaps also can be with reference to described in " molecular cloning experiment guide ".
Then treat measurement information according to acquisition of informations such as the position of marking signal on chip, intensity.If amplified production fluorophor mark also can directly obtain and treat measurement information with fluorescence detection device (such as laser confocal scanning instrument Scanarray3000 etc.).
The labelling groups that sample of nucleic acid is carried out mark includes but not limited to: digoxin molecule (DIG), biotin molecule (Bio), fluorescein and derivative biomolecules (FITC etc.) thereof, other fluorescence molecules (such as Cy3, Cy5 etc.), alkaline phosphatase (AP), horseradish peroxidase (HRP) etc.These marks and marking method thereof all have been routine techniques well-known in the art, also can be with reference to Wang Shenwu chief editor's " gene diagnosis technology-on-radiation operational manual "; J. Pehanorm Brooker, D.W. Russell chief editor, " molecular cloning experiment guide ", Science Press, 2002; Horse stands the people, the Jiang Zhonghua chief editor. biochip. and Beijing: Chemical Industry Press, 2000,1-130.
When above-mentioned sample of nucleic acid and gene chip are hybridized, can first gene chip and prehybridization damping fluid be carried out prehybridization.Solid-phase hybridization between sample of nucleic acid and the gene chip carries out according to the classical way of this area, and the general personnel in this area easily determine relevant damping fluid, probe and the optimum condition of concentration of specimens, prehybridization temperature, hybridization temperature and time etc. according to experience.
Then treat measurement information according to acquisition of informations such as the position of marking signal on gene chip, intensity.If amplified production fluorophor mark also can directly obtain and treat measurement information with fluorescence detection device (such as laser confocal scanning instrument Scanarray3000 etc.).
Detection kit
The present invention also provides a kind of test kit of the expression for detection of described gene.Described test kit can be used for measuring cancerous tissue and the cancer beside organism of (or differentiation) primary hepatocarcinoma.Comprise chip of the present invention in the described test kit.
Preferred, also contain the marker that is useful on the labeled rna sample in the described test kit, and the substrate corresponding with described marker.
In addition, also can comprise in the described test kit for extracting the required all ingredients such as RNA, PCR, hybridization, colour developing, include but not limited to: extract, amplification liquid, hybridization solution, enzyme, contrast liquid, nitrite ion, washing lotion, antibody etc.
In addition, also can comprise working instructions and/or chip image analysis software in the described test kit.Described chip image analysis software is such as the BaiO ArrayDoctor 2.0 of BaiO company, the Arraypro 4.0 of Media Cybernetics company.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is write according to normal condition such as J. Pehanorm Brooker etc. usually, molecular cloning experiment guide, Science Press, the condition described in 2002, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that better implementation method described in the literary composition and material only present a demonstration.
The preparation of embodiment 1, RNA sample
Tissue samples:
The 49 pairs of cancers and cancer beside organism come from the excision sample of Patients with Primary, and these samples come from Qidong liver cancer research institute and the first affiliated hospital of Zhejiang University.10 routine normal hepatocytes come from unexpected death person.Above-mentioned all samples obtain all agreement by the Ethics Committee of WHO cooperative association of Shanghai City government authorization.The clinical data of tissue samples comprises: sex, age, tumor size, pathological grading (Edmonson), liver cirrhosis whether, whether shift, whether recur, HBV and HCV infection conditions etc.All do not accept chemotherapy before all operation in patients, Follow-up After reaches 60 months most.
Gene chip:
Full genomic expression spectrum chip adopts the brilliant core of Boao Biological Co., Ltd (http://www.capitalbio.com)
Human full genome oligonucleotide arrays V1.0 (two channels chip) contains more than 22000 gene probes.
RNA sample (total tissue RNA extraction):
1. the Collection and preservation of sample: in vitro tissue places the liquid nitrogen quick-frozen after cutting into fritter immediately, then moves to-80 ℃ of Refrigerator stores.
2. tissue block is broken: tissue block is put in the liquid nitrogen and pulverizes, and every gram tissue adds 1mlTrizol (Invitrogen company).
3. total RNA extracting: add the chloroform of 0.2ml by every milliliter of Trizol, thermal agitation 15 seconds, incubated at room 2-3 minute; 4 ℃, centrifugal 15 minutes of 10,000g; Colourless supernatant liquor is moved in the new centrifuge tube, add the 0.5ml Virahol, incubated at room 10 minutes, 4 ℃ of 10000g are centrifugal 10 minutes; Outwell supernatant, 0.75ml75% washing with alcohol, 4 ℃, centrifugal 5 minutes of 7,500g; Outwell supernatant, drying at room temperature RNA precipitates 5-10 minute (not making the RNA complete drying), after processing with DEPC without RNA enzyme H2O dissolution precipitation.
4. spectrophotometer standard measure RNA, and the Total RNA that takes a morsel carries out electrophoresis, checks whether RNA degrades.
The gene of embodiment 2, screening significant difference
49 routine cancer samples are used respectively cy3 and two kinds of fluorochrome labels of cy5 with normal sample (10 examples are mixed), the other sample of 49 routine cancers with normal sample (10 examples are mixed), with the probe competitive hybridization on the full genomic expression spectrum chip.The brilliant core of chip after the hybridization
LuxScan
TMThe scanning of 10K micro-array chip scanner obtains result images, and is quantitative to results of hybridization by its LuxScan 3.0 general microarray image analysis softwares that attach at random again.Thus, obtain 49 pairs of cancer samples to normal sample (cancer/normal, Cancer/Normal, C/N) and the other sample of the cancer express spectra data to normal sample (by the cancer/normally, Pericancerous/Normal, P/N).
With by above-mentioned 49 cancers/normal chip results and 49 cancers/normal chip results at first uses LOWESS (locally weighted regression) normalization method.By the cancer after the normalization method/normal (C/N), cancer/normally the chip results of (P/N) is got take the 2 logarithm (log the end of as
2X), use the random Tobin's mean variance model (RVM) after the pairing T method of inspection (because cancerous tissue and cancer beside organism derive from same patient) is proofreaied and correct to screen the significant difference gene that obtains cancer/cancer other (C/P).
For the significance of verifying gene difference is not caused by coincidence, the inventor introduces after the above-mentioned T method of inspection data random rearrangement 1000 times again, detect a gene tested false positive rate (FDR, False Discovery Rate) that is decided to be the significant difference gene after screening by preceding method.
In above-mentioned statistical method, only have the gene of p value＜0.05 just can screenedly be the significant difference gene.
Screening and the checking of sorter (can distinguish two histioid genes)
For seeking the gene that to distinguish two class samples (by cancer and the cancer), the inventor uses the Lib-SVM tool kit of Matlab software, use C-SVC (the support vector sorter of parameters C) and two kinds of methods of V-SVC (the support vector sorter of V parameter), and adopt separately four kinds of kernel function (linear kernel, ploy kernel, gauss kernel, tanh kernel), amount to 8 kinds of methods and make up the svm classifier device.SVM (support vector machine, support vector machine) sorter is the nonlinear function of the logarithmic value of the difference multiple of differential gene between two class samples, its seek can maximize two class sample separation from lineoid, thereby can distinguish best two class samples.
Be the classify accuracy of inspection-classification device, the inventor selects in all crosscheck methods the most stable 10 to take advantage of 10 folding crosscheck methods.10 folding crosscheck methods are that sample totally is divided into 10 sons part, select 1 son part as test data set at every turn, and all the other 9 sons are part as training dataset, repeat 10 times (at every turn with different son parts as test data set).10 assays that so obtain combine and form a assessed value to the sorter classify accuracy.
Again by the highest a kind of as the optimal classification device of selection sort accuracy in 8 kinds of algorithms.
For showing intuitively on the same group similar between sample and not on the same group different between sample, the inventor looks in 3 dimensions and introduces multidimensional scaling (multidimensional scaling, MDS) in the effect.The MDS algorithm for the basis, is determined the position of each sample in lower dimensional space take similarity matrix between the sample-sample that is classified device (one group of gene) definition, and makes it to be adapted to 3 and tie up and look effect.In this 3 dimension space, two samples are more approaching, and then they are more similar; Otherwise, if two samples are at a distance of far away, then more different between them.
The result
Screening during differential gene take p value＜0.05 as the boundary.The cancer sample is to the other sample of cancer (by cancer/cancer, Cancer/Paracancerous, C/P), have 1469 significant difference genes, wherein significantly raise 621 genes that have of (cancer is higher than by the cancer), significantly reduce 848 genes that have of (cancer is lower than by the cancer).
Carrying out sorter when screening, reach the optimal classification precision for guaranteeing to reach the sorter that finds, further select particularly significantly gene of otherness take the p value of differential gene＜0.000001 as the boundary, as the sorter candidate gene.Be inserted in 8 kinds of svm classifier device algorithms and take advantage of 10 crosschecks of rolling over to verify the classify accuracy of sorter with aforesaid 10.After 10 of 1000 secondary data displacement takes advantage of 10 folding cross validations to obtain sorter, for testing the predictive ability of this sorter, 15 samples of inventor's random choose are as unknown sample, with this sorter tissue-derived (by cancer or the cancer) of each sample predicted, observed its prediction accuracy.Comprehensive 8 kinds of algorithms, the classify accuracy that is obtained by this algorithm of C-SVC (gauss kernel, gaussian kernel function) is the highest.33 the genomic constitution sorters (seeing Table 1) that are used for distinguishing cancer and the other sample of cancer that this algorithm obtains.
Table 1: 33 genes that form sorter
For ease of the classifying quality that shows sorter directly perceived, the inventor uses the MDS algorithm 33 gene signal values of each sample to be converted to the eigenvector of 3 dimensions, and they are located in 3 dimension spaces, be depicted as the three-dimensional scatter diagram of the other two class samples of cancer and cancer, see Fig. 1.Can be judged by figure whether the distance between any two samples of different group is larger than the distance between any two samples on the same group.
Embodiment 3, preparation gene detecting chip
The GenBank accession number (GenBank ID) of 33 gene orders that provide according to table 1, the design polynucleotide probes adds the catenation sequence of 0-20nt at the sequence two ends according to features such as the GC that produces sequence compare; Core sequence is different, and catenation sequence is also different.Catenation sequence can be produced at random by program, and the probe that catenation sequence and core sequence form meets the following conditions:
1) in the probe sequence, the quantity of same Nucleotide (A, C, G, T) can not surpass 50% of sequence sum;
2) any continuous A, T or the quantity of C, G can not surpass 25% of sequence sum;
3) G, C content account for the 40%-60% of sequence sum;
4) probe sequence can not be from hybridization, and namely the length of complementary fragment can not surpass 30% of probe length in the probe sequence.
The probe sequence of design sees Table 2.
Table 2
For making stable being combined on the slide of synthetic probe, adopt conventional method to carry out glycosyl modified at 5 ' end of synthetic rear probe.
The point system of chip: first the surface of slide is carried out alkylation and modify, to improve binding ability.Adopt conventional chip point sample method to carry out point sample, in order to detect the repeatability of cross experiment, each probe is at slide point 3-6 hybridization point.
Embodiment 4, test kit preparation
The Chip Packaging of preparation among the embodiment 3 is good, place a box with working instructions, consist of test kit.
The detection of embodiment 5, chip
To sample and the corresponding cancer beside organism sample thereof that obtains 25 routine primary hepatocarcinoma from hospital, press the preparation of embodiment 1 method for the RNA sample that detects, then with the chip (including the probe shown in the SEQ ID NO:1-33 in the table 2 on the chip) of preparation among the embodiment 3, detect with double-blind method.According to the existence of 33 kinds of genes shown in the table 1 whether and the upper downward modulation situation that is in harmonious proportion judge sample to be tested.Wherein, positive control and negative control are respectively known liver cancer tissue sample and cancer beside organism's sample.
The result shows, the sorter that is consisted of by 33 species specific genes, its accuracy can reach 93%, and the predictablity rate of unknown sample is reached 87% (to compare accuracy high with types of organization that hospital is indicated), can very effectively distinguish liver cancer tissue and cancer beside organism.
All quote in this application as a reference at all documents that the present invention mentions, just as each piece document is quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. a polynucleotide collection is characterized in that, described polynucleotide collection comprises the polynucleotide of the following albumen of encoding:
Fiber gelatinized protein 3, CD209 molecule, estrogen receptor 1; the CD5 quasi-molecule, kynuric acid 3-hydroxylase, N-acyltransferase; carry ester gp F, iron is transferred plain antibacterial peptide, GYS2; steroid-5-alpha-reductase enzyme 2 is urged renal cortex element hormone in conjunction with albumen, ethanol dehydrogenase 4; Cytochrome P450 family 2 subfamily C polypeptide 19; Cytochrome P450 family 2 subfamily C polypeptide 8, coagulation factor 9, Cytochrome P450 family 2 subfamily C polypeptide 9; Metallothionein 1 G; phosphoenolpyruvate carboxykinase, the macrophage receptor of one-tenth collagen structure, Metallothionein 1 H; Afamin; glycine-N-acyltransferase, retinol dehydrogenase 16, GNMT; first ammonium second lactone-homocysteine methyl transferase; tyrosine aminotransferase, ApoA5, serine dehydratase; secretor type phosphorprotein 2; L-Ala-oxoethanoic acid transaminase 2 sample albumen 1, Cytochrome P450 family 26 subfamily A polypeptide 1,17-beta hydroxysteroid dehydrogenase 6 homologous proteins and minopontin.
2. polynucleotide collection as claimed in claim 1; it is characterized in that; the described fiber gelatinized protein 3 of encoding; the CD209 molecule; estrogen receptor 1; the CD5 quasi-molecule; kynuric acid 3-hydroxylase; the N-acyltransferase; carry ester gp F, iron is transferred plain antibacterial peptide, GYS2; steroid-5-alpha-reductase enzyme 2; urge renal cortex element hormone in conjunction with albumen, ethanol dehydrogenase 4, Cytochrome P450 family 2 subfamily C polypeptide 19; Cytochrome P450 family 2 subfamily C polypeptide 8; coagulation factor 9, Cytochrome P450 family 2 subfamily C polypeptide 9, Metallothionein 1 G; phosphoenolpyruvate carboxykinase; become the macrophage receptor of collagen structure, Metallothionein 1 H, Afamin; glycine-N-acyltransferase; retinol dehydrogenase 16, GNMT, first ammonium second lactone-homocysteine methyl transferase; tyrosine aminotransferase; ApoA5, serine dehydratase, secretor type phosphorprotein 2; L-Ala-oxoethanoic acid transaminase 2 sample albumen 1; Cytochrome P450 family 26 subfamily A polypeptide 1, the polynucleotide of 17-beta hydroxysteroid dehydrogenase 6 homologous proteins and minopontin are respectively: sequence such as GenBank accession number NM_003665.2, NM_021155.3; NM_021155.3; NM_005894.2, NM_003679.3, NM_000015.2; NM_001638.2; NM_021175.2, NM_021957.3, NM_000348.3; NM_001882.3; NM_000670.3, NM_000769.1, NM_000770.3; NM_000133.3; NM_000771.3, NM_005950.1, NM_002591.3; NM_006770.3; NM_005951.2, NM_001133.2, NM_201648.2; NM_003708.3; NM_018960.4, NM_001713.2, NM_000353.2; NM_052968.3; NM_006843.2, NM_006944.2, NM_031279.2; NM_000783.3, the polynucleotide shown in NM_003725.2 and the NM_001040058.1.
3. the purposes of claim 1 or 2 described polynucleotide collection is characterized in that, for the preparation of the cancerous tissue of measuring primary hepatocarcinoma and detection reagent or the test kit of cancer beside organism.
4. purposes as claimed in claim 3 is characterized in that, described reagent is the primer pair that specific amplification claim 1 or 2 described polynucleotide are concentrated each polynucleotide; Or the probe of specific recognition claim 1 or concentrated each polynucleotide of 2 described polynucleotide.
5. one kind is used for measuring the cancerous tissue of primary hepatocarcinoma and the reagent of cancer beside organism, and it is specific recognition sequence such as GenBank accession number NM_003665.2, NM_021155.3, NM_021155.3, NM_005894.2, NM_003679.3, NM_000015.2, NM_001638.2, NM_021175.2, NM_021957.3, NM_000348.3, NM_001882.3, NM_000670.3, NM_000769.1, NM_000770.3, NM_000133.3, NM_000771.3, NM_005950.1, NM_002591.3, NM_006770.3, NM_005951.2, NM_001133.2, NM_201648.2, NM_003708.3, NM_018960.4, NM_001713.2, NM_000353.2, NM_052968.3, NM_006843.2, NM_006944.2, NM_031279.2, NM_000783.3, the probe of the polynucleotide shown in NM_003725.2 or the NM_001040058.1, the nucleotide sequence of described probe is shown in SEQ ID NO:1-33 is arbitrary.
6. a gene chip is characterized in that, described gene chip comprises:
Solid phase carrier; And
Be fixed in order the probe on the described solid phase carrier, described probe specificity ground is corresponding to sequence such as GenBank accession number NM_003665.2, NM_021155.3, NM_021155.3, NM_005894.2, NM_003679.3, NM_000015.2, NM_001638.2, NM_021175.2, NM_021957.3, NM_000348.3, NM_001882.3, NM_000670.3, NM_000769.1, NM_000770.3, NM_000133.3, NM_000771.3, NM_005950.1, NM_002591.3, NM_006770.3, NM_005951.2, NM_001133.2, NM_201648.2, NM_003708.3, NM_018960.4, NM_001713.2, NM_000353.2, NM_052968.3, NM_006843.2, NM_006944.2, NM_031279.2, NM_000783.3, the polynucleotide shown in NM_003725.2 or the NM_001040058.1.
7. gene chip as claimed in claim 6 is characterized in that, the nucleotide sequence of described probe is shown in SEQ ID NO:1-33 is arbitrary.
8. the purposes of claim 6 or 7 described gene chips is for the preparation of the cancerous tissue of measuring primary hepatocarcinoma and the test kit of cancer beside organism.
9. measure the cancerous tissue of primary hepatocarcinoma and the test kit of cancer beside organism for one kind, it is characterized in that, contain claim 6 or 7 described gene chips in the described test kit.
10. test kit as claimed in claim 9 is characterized in that, also comprises in the described test kit: nucleic acid extract, nitrite ion or hybridization solution.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107653302A (en) * | 2017-11-02 | 2018-02-02 | 安徽师范大学 | For expanding the RT PCR primers and method of female tortoise estrogen receptorαgene |
| CN108615555A (en) * | 2018-04-26 | 2018-10-02 | 山东师范大学 | Colorectal cancer prediction technique and device based on marker gene and mixed kernel function SVM |
| WO2018209639A1 (en) * | 2017-05-18 | 2018-11-22 | 高雄医学大学 | Glycine n-methyl transferase (gnmt) mutant gene and applications thereof |
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| AFFYMETRIX: "Platform GPL570", 《GENE EXPRESSION OMNIBUS》 * |
| DESHUI JIA等: "Genome-Wide Copy Number Analyses Identified Novel Cancer Genes in Hepatocellular Carcinoma", 《HEPATOLOGY》 * |
| SUN LIAO等: "E-GEOD-6222 Transcription profiling of human liver cancers", 《ARRAYEXPRESS》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018209639A1 (en) * | 2017-05-18 | 2018-11-22 | 高雄医学大学 | Glycine n-methyl transferase (gnmt) mutant gene and applications thereof |
| CN107653302A (en) * | 2017-11-02 | 2018-02-02 | 安徽师范大学 | For expanding the RT PCR primers and method of female tortoise estrogen receptorαgene |
| CN108615555A (en) * | 2018-04-26 | 2018-10-02 | 山东师范大学 | Colorectal cancer prediction technique and device based on marker gene and mixed kernel function SVM |
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