CN105441571A - Application of C21orf62 gene in liver cancer diagnosis - Google Patents
Application of C21orf62 gene in liver cancer diagnosis Download PDFInfo
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- C—CHEMISTRY; METALLURGY
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Abstract
The invention discloses application of a C21orf62 gene in liver cancer diagnosis. The C21orf62 gene is in low expression in tissues of a liver cancer patient, through detecting the expression level of the C21orf62 gene, whether a patient suffers from liver cancer can be diagnosed, and through the application of the C21orf62 gene in liver cancer diagnosis, sensitivity and specificity of liver cancer diagnosis are greatly improved.
Description
Technical field
The present invention relates to biological technical field, relate to the purposes of C21orf62 gene in diagnosing cancer of liver particularly.
Background technology
Liver cancer and liver malignancy, can be divided into primary and the large class of Secondary cases two.Characters of Primary Malignant Tumors of Liver originates from epithelium or the mesenchymal tissue of liver, and the former is called primary hepatocarcinoma, is that China is occurred frequently, very harmful malignant tumour; The latter is called sarcoma, comparatively rare compared with primary hepatocarcinoma.Secondary cases or title secondary liver cancer mean that the malignant tumour of the multiple organ origin of whole body is invaded to liver.Liver cancer is one of the most common cancer the highest with grade malignancy, and occurred frequently in Asia and African Territories, the sickness rate of western countries' liver cancer is also raising gradually in recent years.At present, liver cancer has become the second largest cancer killer of China, has very high lethality rate.The cause of disease and the pathogenesis of liver cancer are affirmed not yet completely, may be relevant with the comprehensive action of many factors.Common reason has viral hepatitis, liver cirrhosis etc.5 years survival rates of liver cancer are less than 5%.Liver cancer diagnosis and treatment difficulty, poor prognosis, transfer and relapse is the biggest obstacle affecting liver cancer post-operative survival rates.Generally believing, is the important method improving patient's long-term survival to early detection and the operation of liver cancer person.
Comprise for the method for diagnosing cancer of liver clinically at present: liver cancer serum marker detection, imaging examination; Wherein liver cancer serum marker detection comprises Significance of Determination of Serum Alpha-Fetoprotein and blood zymetology and other tumor markers inspections; Imaging examination comprises ultrasonic examination, CT examination, MRI check, selectivity coeliac artery or hepatic arteriography inspection, the liver puncture hand-manipulating of needle are inhaled cell blood examination and looked into.
Taking the circumstances into consideration to carry out individual comprehensive therapy according to the different steps of liver cancer clinically, is the key improving curative effect; Methods for the treatment of comprises operation, HAL, hepatic arterial chemoembolization, radio frequency, freezing, laser, microwave and the method such as chemotherapy and radiotherapy.Biotherapy, traditional Chinese medical herbal treatment liver cancer also more application.Along with the development of biotechnology, Biological target therapy will play critical effect.Although the gene that liver cancer is correlated with has report more, these genes most are just because the expression of other cancer related genes is lacked of proper care " by product " that cause, and also really occurs relevant to cancer.Research shows, cause the UPSTREAM BINDING FACTOR of these gene differential expressions may play more crucial effect in driving development of cancer, the Clinical symptoms such as the expression of these UPSTREAM BINDING FACTOR and the generation of cancer, development and prognosis have and contact more closely, therefore find that the upstream regulatory factor of cancer has great importance for the treatment of cancer and research.
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 C21orf62 transcription factor that can be used for hepatocarcinoma early diagnosis.Compare the diagnostic method of traditional liver cancer, use gene marker to carry out diagnosing liver cancer and there is promptness, specificity and susceptibility, make patient just can know risk of cancer in early days in cancer, 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 C21orf62 gene and the application of expression product in the product preparing diagnosing liver cancer thereof.
Further, described product carrys out diagnosing liver cancer by the expression level detecting C21orf62 gene and expression product thereof.
Further, the product of the expression level of described detection C21orf62 comprises: carry out detecting the expression level of C21orf62 gene and expression product thereof with the product of diagnosing liver cancer by RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip.
Further, the product of described RT-PCR diagnosing liver cancer at least comprises the primer of a pair specific amplified C21orf62 gene; The product of described real-time quantitative PCR diagnosing liver cancer at least comprises the primer of a pair specific amplified C21orf62 gene; The product of described immunodetection diagnosing liver cancer comprises: the antibody be combined with C21orf62 protein-specific; The product of described in situ hybridization diagnosing liver cancer comprises: with the probe of the nucleic acid array hybridizing of C21orf62 gene; The product of described chip diagnosing liver cancer comprises: protein chip and gene chip; Wherein, protein chip comprises the antibody be combined with C21orf62 protein-specific, and gene chip comprises the probe with the nucleic acid array hybridizing of C21orf62 gene.
Further, 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.
Further, described gene detecting kit comprises SYBRGreen polymerase chain reaction system, primer pair for increase C21orf62 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 liver cancer, described product can carry out diagnosing liver cancer by the expression level detecting C21orf62 gene in liver cancer tissue.
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 C21orf62 gene for detecting C21orf62 gene transcription level; Described protein chip comprises solid phase carrier and is fixed on the specific antibody of the C21orf62 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 C21orf62 gene transcription level; Described protein immunization detection kit comprises the specific antibody of C21orf62 albumen.Wherein, described reagent comprises the reagent used needed in RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip method detection C21orf62 gene expression dose process.Preferably, described reagent comprises primer for C21orf62 gene and/or probe.
Further, described solid phase carrier comprises inorganic carrier and organic carrier, and described inorganic carrier has included but not limited to silicon carrier, glass carrier, ceramic monolith etc.; Described organic carrier comprises polypropylene film, nylon membrane etc.
Further, described gene detecting kit comprises SYBRGreen polymerase chain reaction system, primer pair for increase C21orf62 gene and house-keeping gene; Described SYBRGreen polymerase chain reaction system comprises: PCR damping fluid, dNTPs, SYBRGreen fluorescence dye.
Further, the primer pair sequence of described amplification C21orf62 gene is as follows: forward primer sequence is 5 '-GGCACTGTCTTCTTCTGA-3 '; Reverse primer sequences is 5 '-TGCTGTTCTTCTGACCTT-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 chip can be used for detecting the expression level of the multiple genes (such as, relevant to liver cancer multiple genes) comprising C21orf62 gene.Described protein chip can be used for detecting the expression level of the multiple protein (such as relevant to liver cancer multiple protein) comprising C21orf62 albumen.By being detected by multiple mark with liver cancer simultaneously, the accuracy rate of diagnosing cancer of liver greatly can be improved.
C21orf62 gene of the present invention can be natural or synthetic, or uses the carrier transfection host cell can expressing the DNA fragmentation of C21orf62 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 present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of conventional prokaryotic host cell comprises intestinal bacteria, Bacillus subtilus etc.Conventional eukaryotic host cell comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is eukaryotic cell, as Chinese hamster ovary celI, COS cell etc.
Further, in the present invention, the nucleic acid of C21orf62 albumen or coding C21orf62 albumen can be given by liposome.
Can be DNA, RNA, DNA-RNA mosaic, PNA or other derivative with the probe of the nucleic acid array hybridizing of C21orf62 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 C21orf62 described in the present invention comprises monoclonal antibody, polyclonal antibody.The specific antibody of described C21orf62 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 C21orf62 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, " C21orf62 gene " comprises the polynucleotide of any function equivalent of people C21orf62 gene and people C21orf62 gene.C21orf62 gene comprises and has more than 70% homology with C21orf62 gene (NC_000021.9) DNA sequence dna in current international common core sequence databank GeneBank, and coding identical function protein DNA sequence;
Preferably, the encoding sequence of C21orf62 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 C21orf62 gene is the DNA sequence dna shown in SEQIDNO.1.
In the context of the present invention, C21orf62 gene expression product comprises the partial peptide of people C21orf62 albumen and people C21orf62 albumen.The partial peptide of described C21orf62 albumen contains the functional domain relevant to liver cancer.
" C21orf62 albumen " comprises any function equivalent of C21orf62 albumen and C21orf62 albumen.Described function equivalent comprises C21orf62 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 C21orf62 under high or low stringent condition.
Preferably, C21orf62 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 C21orf62 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 C21orf62 albumen.Peptide or protein with C21orf62 protein fusion are not limited, as long as the fusion rotein of gained retains the biologic activity of C21orf62 albumen.Fusion rotein in the present invention can have various derivative, these derivatives can be but be not limited to multi-form salt, modified outcome etc., as modified on the amino of polypeptide, carboxyl, hydroxyl, sulfydryl, modifier used includes but not limited to polyoxyethylene glycol, dextran etc. again.Available method well known in the art will contain the nucleic acid clone of encoding fusion protein sequence in various expression vector.
C21orf62 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 C21orf62 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.
The modification of aminoacid sequence can be derived from spontaneous mutation or the rear modification of heredity, also can produce by artificial induction's natural gene.
In the context of the present invention, " diagnosing liver cancer " had both comprised and had judged whether experimenter has suffered from liver cancer, also comprised and judge whether experimenter exists the risk suffering from liver cancer.
Advantage of the present invention and beneficial effect:
Late Cambrian of the present invention C21orf62 genetic expression is relevant to liver cancer, by detecting the expression of C21orf62 in experimenter's liver cancer tissue, can judge whether experimenter suffers from liver cancer or judge whether experimenter exists the risk suffering from liver cancer, thus instruct clinicist to provide prevention scheme or treatment plan to experimenter.
Present invention finds a kind of new molecular marked compound-C21orf62 gene of liver cancer, compare traditional detection means, gene diagnosis more in time, more special, sensitiveer, the early diagnosis of liver cancer can be realized, thus reduce the mortality ratio of liver cancer.
Accompanying drawing explanation
Fig. 1 display utilizes QPCR to detect the expression of C21orf62 gene in liver cancer tissue;
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 liver cancer
1, sample collection
The routine healthy tissues of each collection 8 and liver cancer tissue sample, above-mentioned all samples obtain all by the agreement of the council of organizational ethics, collect after sample frozen in liquid nitrogen.
2, the preparation (utilizing the RNA extraction test kit of organizing of QIAGEN to operate) of RNA sample
1) pre-treatment of sample: take out sample and put into and spend the night and the homogenizer of sterilizing with 0.1%DEPC water soaking from-80 DEG C of liquid nitrogen containers, firmly grind into fine powder; Must make sample all the time in liquid nitrogen environment in process of lapping, the necessary precooling of grinding cup, grinding limit, limit adds liquid nitrogen.
2) cracking: add 1mlTrizolRNA extracting solution in homogenizer, homogenate 10min under room temperature, then leave standstill 10min, allow the abundant cracking of nucleoprotein.
3) purifying: allow the supernatant liquid after homogenate move to in the water-treated aseptic EP pipe of 1.5ml of 0.1%DEPC, put upside down mixing 10s, room temperature leaves standstill 5min, and carries out mark respectively.
4) layering: in the ratio of 0.2:1 (chloroform: Trizol), adds 0.2ml chloroform respectively in each EP pipe, acutely put upside down mixing 15s, then room temperature leaves standstill 5min.
5) centrifugal by putting into 4 DEG C of refrigerated centrifuges in each EP pipe, the centrifugal 10min of 12000rpm.Sample is divided into three layers: yellow organic phase, middle layer, the aqueous phase that upper strata is colourless, RNA, mainly at aqueous phase, transfers to aqueous phase in new EP pipe, carries out mark.Note will being drawn onto middle layer and orlop scarcely, avoid polluting.
6) add isopyknic Virahol, mix under room temperature, and hatch 10min;
7) precipitate: put into 4 DEG C of refrigerated centrifuges more centrifugal, the centrifugal 15min of 12000rpm.
8) wash: abandon supernatant liquor after centrifugal, then add the washing precipitation gently of 1ml75% ethanol.
9) abandon supernatant liquor, blot ethanol in pipe, in room temperature in air, dry RNA precipitates 5-10min.
10) dissolve: add 20 μ lRNasefree deionized water dissolving RNA.
11) detect RNA purity and concentration with UV-light protractor, residue RNA sample is placed in-80 DEG C of Refrigerator stores.
3, high-throughput transcript profile order-checking
1) the RNA-seq section of reading location
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.
2) transcript abundance assessment
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.
3) detection of difference expression gene
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 C21orf62 gene in liver cancer tissue is significantly lower than the expression amount in healthy tissues.
The differential expression of embodiment 2QPCR sequence verification C21orf62 gene
1, large sample QPCR checking is carried out to C21orf62 gene differential expression.According to the sample collection way selection liver cancer tissue in embodiment 1 and each 60 examples of healthy tissues.
2, QPCR concrete operation step is as follows:
(1) RNA extracts
Frozen in liquid nitrogen after collecting sample, organizing the mortar putting into precooling to grind, after tissue samples is powdered after taking-up:
1) add Trizol, room temperature places 5min;
2) add chloroform 0.2ml, use forced oscillation centrifuge tube, fully mix, ambient temperatare puts 5-10min;
3) the centrifugal 15min of 12000rpm, moves on to upper water mutually in another new centrifuge tube and (notes the proteic substance be not drawn onto between two-layer aqueous phase), add the Virahol of isopyknic-20 DEG C of precoolings, fully put upside down mixing, be placed in 10min on ice;
4) 12000rpm carefully discards supernatant liquor at a high speed after 15min, adds 75%DEPC washing with alcohol precipitation (4 DEG C of preservations), washing precipitate in the ratio of 1ml/mlTrizol, vibration mixing, 4 DEG C, the centrifugal 5min of 12000rpm;
5) discard ethanol liquid, ambient temperatare puts 5min, adds DEPC water dissolution precipitation;
6) RNA purity and concentration is measured with Nanodrop2000 ultraviolet spectrophotometer, frozen in-70 DEG C of refrigerators.
(2) reverse transcription
Adopt 25 μ l reaction systems, each sample gets 1 μ g total serum IgE as template ribonucleic acid, adds following component respectively: DEPC water, 5 × RT Buffer, 10mMdNTP, 0.1mMDTT in PCR pipe, 30 μMs of OligodT, 200U/ μ lM-MLV, template ribonucleic acid.Hatch 1h for 42 DEG C, 72 DEG C of 10min, of short duration centrifugal.
(3) QPCR amplification inspection
Design of primers
The primer sequence of C21orf62 gene is:
Forward primer: 5 '-GGCACTGTCTTCTTCTGA-3 ' (SEQIDNO.3)
Reverse primer: 5 '-TGCTGTTCTTCTGACCTT-3 ' (SEQIDNO.4)
The primer sequence of house-keeping gene GAPDH is:
Forward primer: 5 '-CTCTGGTAAAGTGGATATTGT-3 ' (SEQIDNO.5)
Reverse primer: 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQIDNO.6)
Adopt 25 μ l reaction systems, each sample arranges 3 parallel pipes, and all amplified reactions are above to ensure the reliability of result all in triplicate.Prepare following reaction system: SYBRGreen polymerase chain reaction system 12.5 μ l, forward primer (5 μMs) 1 μ l, reverse primer (5 μMs) 1 μ l, template cDNA2.0 μ l, without enzyme water 8.5 μ l.Operations is all in carrying out on ice.Amplification program is: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 45s) × 30 circulations.Using SYBRGreen as fluorescent marker, in the enterprising performing PCR reaction of LightCycler fluorescence real-time quantitative PCR instrument, by melt curve analysis analysis and electrophoresis determination object band, Δ Δ CT method carries out relative quantification.
3, statistical method
Experiment has all come for 3 times according to repetition, result data is all represent in the mode of mean+SD, adopt 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.
4, result
Result as shown in Figure 1, compared with healthy tissues, lower at Expression In Hepatocellular Carcinoma by C21orf62 gene, and difference has statistical significance (P<0.05), 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)
- The application in the product preparing diagnosing liver cancer of 1.C21orf62 gene and expression product thereof.
- 2. application according to claim 1, is characterized in that, described product carrys out diagnosing liver cancer by the expression level detecting C21orf62 gene and expression product thereof.
- 3. application according to claim 2, it is characterized in that, the product of the expression level of described detection C21orf62 comprises: carry out detecting the expression level of C21orf62 gene and expression product thereof with the product of diagnosing liver cancer by RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip.
- 4. application according to claim 3, is characterized in that, the product of described RT-PCR diagnosing liver cancer at least comprises the primer of a pair specific amplified C21orf62 gene; The product of described real-time quantitative PCR diagnosing liver cancer at least comprises the primer of a pair specific amplified C21orf62 gene; The product of described immunodetection diagnosing liver cancer comprises: the antibody be combined with C21orf62 protein-specific; The product of described in situ hybridization diagnosing liver cancer comprises: with the probe of the nucleic acid array hybridizing of C21orf62 gene; The product of described chip diagnosing liver cancer comprises: protein chip and gene chip; Wherein, protein chip comprises the antibody be combined with C21orf62 protein-specific, and gene chip comprises the probe with the nucleic acid array hybridizing of C21orf62 gene.
- 5. the application according to any one of claim 1-4, 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.
- 6. application according to claim 5, is characterized in that, described gene detecting kit comprises SYBRGreen polymerase chain reaction system, primer pair for increase C21orf62 gene and house-keeping gene; Described SYBRGreen polymerase chain reaction system comprises: PCR damping fluid, dNTPs, SYBRGreen fluorescence dye.
- 7. a product for diagnosing liver cancer, is characterized in that, described product can carry out diagnosing liver cancer by the expression level detecting C21orf62 gene in liver cancer tissue.
- 8. product according to claim 7, 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.
- 9. product according to claim 8, is characterized in that, described gene detecting kit comprises SYBRGreen polymerase chain reaction system, primer pair for increase C21orf62 gene and house-keeping gene; Described SYBRGreen polymerase chain reaction system comprises: PCR damping fluid, dNTPs, SYBRGreen fluorescence dye.
- 10. product according to claim 8, is characterized in that, the primer pair sequence of described amplification C21orf62 gene is as follows: forward primer sequence is 5 '-GGCACTGTCTTCTTCTGA-3 '; Reverse primer sequences is 5 '-TGCTGTTCTTCTGACCTT-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 '.
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Cited By (1)
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| CN107164536A (en) * | 2017-07-07 | 2017-09-15 | 邳州东大医院 | Application of the MRNIP genes in major depressive disorder diagnosis |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107164536A (en) * | 2017-07-07 | 2017-09-15 | 邳州东大医院 | Application of the MRNIP genes in major depressive disorder diagnosis |
| CN107164536B (en) * | 2017-07-07 | 2019-06-11 | 邳州东大医院 | Application of the MRNIP gene in major depressive disorder diagnosis |
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