CN110117656A - A kind of biomarker relevant to sdenocarcinoma of stomach occurrence and development - Google Patents

Abstract

The invention discloses a kind of biomarkers relevant to sdenocarcinoma of stomach occurrence and development, the present invention has found that RP11-320G24.1 expresses up-regulation in patients with gastric cancer by high throughput sequencing technologies combination bioinformatic analysis for the first time, and further demonstrating RP11-320G24.1 by QPCR is difference expression gene, and RP11-320G24.1 is prompted to can be used as the diagnosing and treating that biomarker is applied to gastric cancer.

Description

A kind of biomarker relevant to sdenocarcinoma of stomach occurrence and development

Technical field

The invention belongs to biomedicine fields, are related to a kind of biomarker relevant to sdenocarcinoma of stomach occurrence and development.

Background technique

Gastric cancer (gastric cancer, GC) is a kind of malignant tumour common in digestive system, there is very high morbidity Rate and case fatality rate, annual new hair gastric cancer cases about 1,000,000 in the whole world, seriously threaten human health and quality of life.Although now We achieve some progress in terms of the clinical treatment of gastric cancer and basic research, but many factors participate in gastric cancer and send out Exhibition, pathogenesis is sufficiently complex, still needs to be illustrated there are many problem.Find the important molecule of Effective Regulation gastric cancer occurrence and development It can instruct to research and develop clinical gastric cancer molecular diagnosis and molecular targeted agents, it is also necessary to which further investigation is inquired into.

Human genome sequencing result analysis shows, 70%-90% gene is transcribed in full-length genome, but only less than 2% gene is finally translated as albumen, remaining overwhelming majority transcripts be named as non-coding RNA (non-coding RNA, NcRNA) (TayY, Rinn J, Pandolfi PP.The multilayered complexity of ceRNA crosstalk and competition.Nature.2014；505(7483):344-52.).Wherein containing small less than 200 nucleotide RNA (microRNA, miRNA) and greater than 200 nucleotide long-chain non-coding RNA (long non-coding RNA, 1ncRNA).Currently, having large result about miRNA functional study, 1ncRNA also becomes research hotspot now.

1ncRNA, most transcribed by rna plymerase ii (RNA polymerase II, RNAPII) generate, and cannot encode egg It is white, but biological function is extremely complex, many levels such as adjustable transcription, translation, epigenetic modification.It has now been found that 1ncRNA participates in the occurrence and development of a variety of diseases, and the regulating and controlling effect to tumour is the hot spot studied now.Research is found The controllable kinds of tumors including lung cancer, gastric cancer, liver cancer, breast cancer, prostate cancer of 1ncRNA.Although emerging in recent years Research and Relational database of many about 1ncRNA, but the functional mechanism of most 1ncRNA is not still by mankind institute Understand.1ncRNA is a very promising research direction.The relationship for studying gastric cancer and lncRNA, for realizing the essence of gastric cancer Standardization diagnosis and personalized treatment have great importance.

Summary of the invention

In order to make up for the deficiencies of the prior art, an object of the present invention provides a kind of detection long-chain non-coding RNA The product of RP11-320G24.1, to realize that the early diagnosis of gastric cancer provides basis.

The second object of the present invention provides a kind of diagnostic method of gastric cancer, by detecting lncRNA RP11-320G24.1 Expression diagnose whether patient suffers from gastric cancer.

To achieve the goals above, the present invention adopts the following technical scheme:

The present invention provides the reagents of detection long-chain non-coding RNA RP11-320G24.1 in the product for preparing diagnosis of gastric cancer In application.

Wherein, RP11-320G24.1 includes RP11-320G24.1 gene and its homologue, mutation and isoform.The art Language covers overall length, unprocessed RP11-320G24.1, and from any type of RP11-320G24.1 processed in cell. The term covers the natural generation variant (such as splice variant or allelic variant) of RP11-320G24.1.The term is covered for example RP11-320G24.1 gene, the gene order of RP11-320G24.1 and come from any other vertebrate origin.It is public at present There are two transcripts by the RP11-320G24.1 opened: ENST00000452690.1 and ENST00000447570.1.In the present invention Specific embodiment in, a kind of representative RP11-320G24.1 sequence is as shown in ENST00000452690.1.

Those skilled in the art will be seen that, when carrying out bioinformatic analysis to sequencing result, it will usually will Sequencing result and known genome are compared, as long as sequencing fragment can compare on relevant gene, so that it may regard as It is the expression of the gene, therefore, the different transcription products of RP11-320G24.1 are also contained in the present invention.

Further, RP11-320G24.1 expresses up-regulation in patients with gastric cancer.

Further, the product includes: by sequencing technologies, nucleic acid hybridization technique, nucleic acid amplification technologies or immunoassays Method detection RP11-320G24.1 gene expression.

Further, the nucleic acid amplification technologies are selected from polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), the amplification (TMA) of transcriptive intermediate, ligase chain reaction (LCR), strand displacement amplification (SDA) and be based on nucleic acid sequence The amplification (NASBA) of column.

In general, PCR uses the annealing of denaturation, primer pair and opposite strand and multiple circulations of primer extend, with index side The copy number of formula increase target nucleic acid sequence；Reverse transcriptase (RT) is then used for the DNA (cDNA) complementary from mRNA preparation by RT-PCR, Then cDNA is generated to multiple copies of DNA by PCR amplification；TMA is in substantially constant temperature, ionic strength and pH Under the conditions of autocatalytically synthesize multiple copies of target nucleic acid sequence, wherein multiple RNA copy of target sequence is autocatalytically given birth to At other copy, TMA is optionally included using blocking, part, terminate part and other modified parts, to improve TMA process Sensitivity and accuracy；LCR uses the two groups of complementary DNA oligonucleotides hybridized with the adjacent area of target nucleic acid.DNA few nucleosides Acid is covalently attached in thermal denaturation, hybridization and the multiple circulations of the repetition of connection by DNA ligase, to generate detectable double-strand Connect oligonucleotide product；SDA uses multiple circulations of following steps: primer sequence pair and the opposite strand of target sequence move back Fire carries out primer extend under there are dNTP α S to generate (hemiphosphorothioated) of half thiophosphorylation of double-strand Primer extension product, the nicking that the endonuclease that semi-modified restriction enzyme enzyme recognition site carries out mediates, and from cutting The polymerase-mediated object drawn that the mouth end 3' carries out extends to replace existing chain and generate and set for next round primer annealing, nicking and chain The chain changed expands so as to cause the geometry of product.

It is described the present invention provides a kind of product of lncRNA RP11-320G24.1 expression in vitro detection sample Product includes chip, kit or nucleic acid film item." sample " includes cell, tissue or body fluid.Body for use in the present invention Liquid includes blood, lymph, urine, saliva or any other body secretion fluid or derivatives thereof.Blood may include whole blood, blood Slurry, serum or any blood derivatives.Preferably, the sample is tissue, blood.In a specific embodiment of the invention, Selected sample is tissue.

Further, the chip, kit or nucleic acid film item include for RP11-320G24.1 specific primer pair or Probe.

Further, the specific primer to for SYBR Green, Taqman probe, molecular beacon, double cross probe, The detection of combined probe.

Further, the sequence of the specific primer pair is as shown in SEQ ID NO.1 and SEQ ID NO.2.

The present invention provides a kind of application of product recited above in the tool for preparing diagnosis of gastric cancer.

The present invention provides application of the RP11-320G24.1 in the computation model of building prediction gastric cancer.

It can be appreciated that, the measurement of two or more markers can be used to improve investigation as those of skill in the art In diagnosis problem.Biochemical marker can measure individually, or in one embodiment of the invention, they can be simultaneously Measurement, such as using chip or based on the array technique of pearl.Then the independent concentration for interpreting biomarker, such as use every kind Individual retentions of marker or their combinations are interpreted.

In the present invention, it can be implemented in various ways marker levels and certain possibility or risk association and realize The step of getting up.Preferably, the mathematically measurement concentration of combination gene and one or more other markers, and by combined value It associates with basic diagnosis problem.It can be by any suitable prior art mathematical method by the measurement group of marker levels It closes.

Preferably, the mathematical algorithm applied in marker combination is a kind of logarithmic function.Preferably, using such mathematics Algorithm or such logarithmic function the result is that single value.According to basic diagnosis problem, can easily by such value with it is for example a Body associates about the risk of gastric cancer or with the other intentional diagnostic uses for helping to assess gastric cancer pine patient.With a kind of preferred Mode, such logarithmic function obtains as follows: individual segregation a) is entered into group, such as normal person, have gastric cancer risk individual, Patient etc. with gastric cancer, b) marker of the significant difference between these groups, c are identified by univariate analysis) logarithm Regression analysis d) constructs logarithmic function to assess the independent difference value that can be used for assessing these difference groups of marker to combine Independent difference value.In such analysis, marker is no longer independent, but represents a marker combination.

Logarithmic function for marker combination to be got up with disease association is preferably developed using by applied statistical method With the algorithm of acquisition.For example, suitable statistical method is discriminant analysis (DA) (i.e. linear, secondary, regular DA), Kernel method (i.e. SVM), nonparametric technique (i.e. k- nearest neighbor classifiers), PLS (partial least square), method (the i.e. logic based on tree Recurrence, CART, random forest method, boosting/bagging method), generalized linear model (i.e. logarithm regression), the side based on principal component Method (i.e. SIMCA), broad sense Additive Model, the method based on fuzzy logic, the method based on artificial neural network and genetic algorithms.Skillfully Technical staff merges in the suitable statistical method of selection to assess marker group of the invention thus to obtain suitable mathematical algorithm Aspect will not be problematic.In one embodiment, for obtaining the statistical method choosing of mathematical algorithm used in assessment gastric cancer From DA (i.e. linear, secondary, rule based judgment analysis), Kernel method (i.e. SVM), (i.e. k- nearest-neighbors are classified for nonparametric technique Device), PLS (partial least square), the method (i.e. logistic regression, CART, random forest method, propelled method) based on tree or Generalized linear model (i.e. logarithm regression).

The present invention provides application of the RP11-320G24.1 in the pharmaceutical composition of preparation treatment gastric cancer.

Further, the drug includes the reagent for lowering RP11-320G24.1 expression and/or pharmaceutically acceptable load Body.

Terms used herein " differential expression " indicates to mark with one or more present invention biologies identical in second of sample The expression of will object compares, after measured the amount or level of RNA, one or more biological markers of the invention in a sample The difference of one or more splice variant expressions of the RNA of the object and/or biomarker RNA." differential expression " It can also include compared with protein expression quantity in second of sample or sample group or level, to of the invention in sample or sample group The measurement of the protein of biomarker coding.Differential expression can it is as described herein and those skilled in the art understand that method It determines.Term " differential expression " or " variation of expression " indicate and give measuring for biomarker in second of sample Expression compares, after measured the amount of RNA and/or the amount of protein, and biomarker is given in sample can measure expression Increase or decrease.Term " differential expression " or " variation of expression " also may indicate that marks with biology in second sample group The expression that measures of will object compares, and biomarker is given in sample group can measure increasing or decreasing for expression.This " differential expression " used in text can be with the expression of given biomarker relative to the flat of biomarker given in control The ratio of equal expression is measured, and wherein ratio is not equal to 1.0.Differential expression can also be measured with p value.When use p value When, when p value is less than 0.1, biomarker is accredited as the differential expression between the first and second groups.More preferable p value is less than 0.05.Even more preferably p value is less than 0.01.Even more preferably from p value less than 0.005.Most preferably p value is less than 0.001.Work as sketch-based user interface When determining differential expression, if the ratio of expression is more than or less than 1.0, RNA or egg in the first and second of sample White matter is differential expression.For example, the ratio greater than 1.2,1.5,1.7,2,3,4,10,20, or the ratio less than 1, example Such as 0.8,0.6,0.4,0.2,0.1,0.05.In another embodiment of the present invention, if the average expression of the first group The horizontal ratio with the Average expression level of the second group is more than or less than 1.0, then transcribed nucleic acid is originally differential expression.Citing For, the ratio greater than 1.2,1.5,1.7,2,3,4,10,20, or the ratio less than 1, such as 0.8,0.6,0.4,0.2,0.1, 0.05.In another embodiment of the present invention, if average table in expression and second colony in first sample Up to horizontal ratio be more than or less than 1.0, for example including ratio be greater than 1.2,1.5,1.7,2,3,4,10,20 or ratio be less than 1, such as 0.8,0.6,0.4,0.2,0.1,0.05, then transcribed nucleic acid is originally differential expression.

" differential expression increase " or " up-regulation " indicates gene relative in contrast, and gene expression is (with rna expression or albumen Matter expression measurement) display increase at least 10% or more, such as 20%, 30%, 40% or 50%, 60%, 70%, 80%, 90% or more or 1.1 times, 1.2 times, 1.4 times, 1.6 times, 1.8 times or more.In a specific embodiment of the invention, gene Expression refers to rna expression.

" differential expression reduction " or " downward " indicates gene relative in contrast, and gene expression is (with rna expression or albumen Matter expression measurement) display reduce at least 10% or more, such as 20%, 30%, 40% or 50%, 60%, 70%, 80%, 90% or less than 1.0 times, 0.8 times, 0.6 times, 0.4 times, 0.2 times, 0.1 times or less gene.In specific implementation of the invention In mode, gene expression refers to rna expression.

In the present invention, " probe ", which refers to, to divide in conjunction with the particular sequence of another molecule or subsequence or other parts Son.Unless otherwise indicated, term " probe " is often referred to match and another polynucleotides (often referred to as " target by complementary base Polynucleotides ") combine polynucleotide probes.According to the preciseness of hybridization conditions, probe energy and lack complete sequence with the probe The complementary target polynucleotide of column combines.Probe can make direct or indirect label, and range includes primer.Crossing system, packet It includes, but is not limited to: solution phase, solid phase, mixed phase or in situ hybridization measuring method.

The probe has the base sequence with the specific base sequence complementary of target gene.Here, so-called " complementation ", As long as hybridization, can not be complete complementary.These polynucleotides have usually relative to the specific base sequence 80% or more, preferably 90% or more, more preferable 95% or more, particularly preferred 100% homology.These probes can be DNA, It is also possible to RNA, furthermore it is possible to pass through PNA (Polyamide nucleic in part of it or whole nucleotides Acid, peptide nucleic acid), LNA (registered trademark, locked nucleic acid, Bridged Nucleic Acid, Cross-linked core Acid), ENA (registered trademark, 2 '-O, 4 '-C-Ethylene-bridged nucleic acids), GNA (Glycerol Nucleic acid, glycerol nucleic acid), the artificial replacement nucleic acid such as TNA (Threose nucleic acid, threose nucleic acid) obtains Polynucleotides.

Term " homology " refers to complementary degree.There may be Homoeologies or complete homology (that is, same Property).The sequence of partial complementarity is at least partly to inhibit the nucleic acid molecules of complete complementary and " substantially homologous " target nucleic acid miscellaneous The nucleic acid molecules of friendship.Fully-complementary sequence can be by being surveyed under the conditions of property low strict using hybridization with the inhibition of target sequence hybridized Fixed (Southern or Northern trace, solution hybridization etc.) is checked.Substantially homologous sequence or probe will compete simultaneously Inhibit complete homologous nucleic acid molecules under the conditions of property low strict and the combination (that is, hybridization) of target.This is not to say, low strict Property condition is to allow the condition of non-specific binding；Spy is combined between two sequences of property condition requirement low strict The interaction of anisotropic (that is, selectivity).Being not present for non-specific binding can be by using substantially incomplementarity (for example, low In about 30% identity) the second target tested；In the case where non-specific binding is not present, probe will not be with second Incomplementarity target hybridization.

Term " hybridization " in the present invention is used to refer to the pairing of complementary nucleic acid.Hybridization and intensity for hybridization are (that is, between nucleic acid Association intensity) influenced by factor such as below: the stringency of complementarity, related condition between nucleic acid is formed Hybrid Tm and nucleic acid in G:C ratio.The individual molecule of pairing in its structure containing complementary nucleic acid is known as " self Hybridization ".

Gene detecting kit or genetic chip can be used for detecting including RP11-320G24.1 gene in the present invention The expression of multiple genes (for example, multiple genes relevant to gastric cancer) detects multiple markers of gastric cancer simultaneously, It is greatly improved the accuracy rate of diagnosing gastric cancer.

It would be recognized by those skilled in the art that practicability of the invention is not limited to marker gene of the invention The gene expression of any specific variants is quantified.

Nucleic acid hybridization technique in the present invention include but is not limited in situ hybridization (ISH), microarray and Southern or Northern trace.In situ hybridization (ISH) be it is a kind of use label complementary DNA or RNA chain as probe with position tissue one Part or slice (original position) or if organize it is sufficiently small if for entirely organize (full organization embedding ISH) in specific DNA or The hybridization of RNA sequence.DNA ISH can be used for determining the structure of chromosome.RNA ISH is for measuring with position tissue slice or entirely MRNA and other transcripts (for example, ncRNA) in organization embedding.Usually sample cell and tissue are handled in situ solid Targeting transcript, and increase the entrance of probe.Probe hybridizes with target sequence at high temperature, then washes off extra probe.Point Not Shi Yong autoradiograph, fluorescence microscopy or immunohistochemistry, in tissue with radiation, fluorescence or antigenic mark base The probe of label is positioned and is quantified.ISH can also be used two or more to pass through radioactivity or other nonradioactive labelings The probe of substance markers, to detect two or more transcripts simultaneously.

Term " microarray ", including but not limited to: DNA microarray (for example, cDNA microarray and oligonucleotide microarray), Protein microarray, micro-array tissue, transfection or cell microarray, chemical compound microarray and Antibody microarray.Commonly referred to as DNA microarray for genetic chip, DNA chip or biochip is the set of microcosmic DNA point, these points are connected to the surface of solids On (for example, glass, plastics or silicon chip), formed for several thousands genes to be carried out with expression pattern analysis or expression prison simultaneously The array of survey.Fixed DNA fragmentation is known as probe, thousands of to can be used in single DNA microarray.Microarray can be used for passing through Compare the gene expression in disease and normal cell and identifies disease gene or transcript (for example, ncRNA).Microarray can be used Multiple technologies are manufactured, including but not limited to: be printed on glass slide with apicule needle, carried out using prefabricated mask photoetching, The electrochemical method on photoetching, ink jet printing or microelectrode array is carried out using dynamic micro mirror element.

Southern and Northern trace is respectively used to detection specific DNA or RNA sequence.Make to extract from sample DNA or RNA fracture, it is separated by electrophoresis on matrix gel, be then transferred on molecular filter.Make filter combine DNA or RNA with and the complementary label probe of sequence of interest hybridize.Detection is integrated to the hybridization probe of filter.A kind of change of the program Change form is reverse northern trace, wherein the substrate nucleic acid for being fixed to film is the set of isolated DNA fragmentation, and probe is From tissue extraction and the RNA that is marked.

Term " diagnosis " in the present invention refers to through the S&S of disease or genetic analysis, pathological analysis, tissue The identification disease such as credit analysis.

The advantages of the present invention:

Present invention finds a kind of molecular markers of diagnosis of gastric cancer, by the table for detecting marker RP11-320G24.1 Early carcinoma of stomach can be judged up to level, to carry out early intervention, improve the survival rate of patient.

The present invention provides a kind of and closely related genes of gastric cancer occurrence and development, provide for the scientific research of gastric cancer Theoretical foundation.

Detailed description of the invention

Fig. 1 is the expression figure using QPCR detection RP11-320G24.1 gene in stomach organization.

Specific embodiment

The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip Part, or according to the normal condition proposed by manufacturer.

Embodiment 1 screens gene marker relevant to gastric cancer

1, sample collection

The cancerous tissue and corresponding cancer beside organism's sample of 4 sdenocarcinomas of stomach are collected, high-flux sequence, all patient's arts are carried out It is preceding not carry out chemotherapy, radiotherapy and endocrine therapy.

2, the preparation and quality analysis of RNA sample

The extraction of total serum IgE, step are carried out using the total RNA from animal tissues extracts kit (catalog number (Cat.No.) DP431) of Tiangeng It is detailed in specification.

1) homogenized

Every 10-20mg tissue plus 300 μ l lysate RL are thoroughly ground tissue with grinding pestle；Add then in homogenate Enter 590 μ l RNase-Free ddH₂O and 10 μ l Proteinase K, 56 DEG C of processing 10-20min after mixing.

2) 12,000rpm is centrifuged 2-5min, and supernatant is taken to perform the following operation.

3) it is slowly added to 0.5 times of supernatant volume dehydrated alcohol, is mixed, obtained solution and precipitating is transferred to adsorption column together In CR3 (adsorption column is placed in collecting pipe), 12,000rpm centrifugation 30s discard the waste liquid in collecting pipe, adsorption column are put back to receipts In collector.

4) 350 μ l protein liquid removal RW1,12,000rpm centrifugation 30s are added into adsorption column CR3 and waste liquid are abandoned, by adsorption column It puts back in collecting pipe.

5) the DNase I working solution of 80 μ l is added to the center adsorption column CR3, is placed at room temperature for 15min.

6) 350 μ l protein liquid removal RW1,12,000rpm centrifugation 30s are added into adsorption column CR3 and waste liquid are abandoned, by adsorption column It puts back in collecting pipe.

7) 500 μ l rinsing liquid RW are added into adsorption column CR3, are stored at room temperature 2min, 12,000rpm centrifugation 30s are abandoned useless Liquid puts back to adsorption column CR3 in collecting pipe.

8) step 7) is repeated.

9) 12,000rpm is centrifuged 2min, outwells waste liquid.Adsorption column CR3 is placed in and is placed at room temperature for several minutes, thoroughly to dry Remaining rinsing liquid in adsorbent material.

10) adsorption column CR3 is transferred in a new RNase-Free centrifuge tube, is vacantly dripped to the intermediate position of adsorbed film Add 30-100 μ l RNase-Free ddH₂O, is placed at room temperature for 2min, and 12,000rpm centrifugation 2min obtain RNA solution.

11) quality testing of RNA

With the integrality (deposition condition: gum concentration 1.2% of agarose gel electrophoresis detection RNA；0.5 × TBE running buffer Liquid；150V, 15min) detection integrality.When 28S rRNA is twice of 18S rRNA, illustrate that the integrality of RNA is preferable.

With the concentration and purity of spectrophotometer detection RNA, OD260/OD280 is read between 1.8 and 2.1, the matter of RNA It measures higher.

3, the building and sequencing of cDNA library

The building and sequencing of cDNA library are completed by Hua Da gene, and steps are as follows:

1) total serum IgE DNase I digests: using DNA fragmentation present in DNase I digestion Total RNA sample, magnetic bead is pure Change recycling reaction product, is finally dissolved in DEPC water；

2) rRNA: the good Total RNA sample of cancellationization is removed, is removed using the Ribo-Zero kit of Epicentre RRNA carries out Agilent 2100 after removal and detects, verifies rRNA removal effect；

3) RNA is interrupted: taking previous step sample, addition interrupts Buffer, is placed in progress heat in PCR instrument and interrupts, interrupts 140-160nt；

4) synthesis of one chain of reverse transcription: appropriate primer being added into the sample after interrupting, after mixing well Thermomixer thermophilic reacts certain time, is allowed to open secondary structure and in conjunction with primer, adds the chain prepared in advance Synthetic reaction system Mix synthesizes a chain cDNA by corresponding program in PCR instrument；

5) synthesis of two chain of reverse transcription: preparing two chain synthesis reaction systems, one timing of thermophilic reaction on Thermomixer Between, synthesis has the two chain cDNA of dUTP, and reaction product carries out purification and recovery with magnetic bead；

6) end is repaired: being prepared end and is repaired reaction system, thermophilic reacts certain time in Thermomixer, in enzyme Under the action of, the cohesive end for the cDNA double-strand that reverse transcription obtains is repaired, end reparation product is purified with magnetic bead Recycling, is finally dissolved in EB Solution for sample；

7) 3 ' end cDNA adds " A ": it prepares and adds " A " reaction system, thermophilic reacts certain time in Thermomixer, Under the action of enzyme, 3 ' ends for the product cDNA for repairing end are plus A base；

8) connection of cDNA5 ' adapter: preparing connector coupled reaction system, the thermophilic reaction one in Thermomixer It fixes time, under the action of enzyme, connect connector with A base, product carries out purification and recovery with magnetic bead；

9) UNG digests bis- chain of cDNA: UNG digestion reaction system is prepared, two chains in double-stranded DNA are fallen by UNG enzymic digestion, And purification and recovery is carried out to product with magnetic bead；

10) PCR reaction and product recycling: PCR reaction system is prepared, PCR response procedures appropriate is selected, upper step is obtained To product expanded, to PCR product carry out magnetic beads for purifying recycling, recovery product is dissolved in EB solution, labelled.

11) Library Quality detects: using Agilent 2100Bioanalyzer and ABI StepOnePlus Real- Time PCR System detects Library Quality；

12) machine is sequenced on: detecting qualified library, NaOH denaturation is added at single-stranded, it is anticipated that upper machine data volume, dilution To certain upper machine concentration.Library after denaturation dilution is added in FlowCell, is hybridized with the connector on FlowCell, Bridge-type PCR amplification is completed on cBot, is finally sequenced using Illumina Hiseq x-ten platform.

4, bioinformatic analysis

1) with cutadapt to the 5 ' of reads and 3 ' Duan Jinhang trim, trim falls the base of quality < 20, and it is big to delete N In 10% reads；

2) tophat is compared onto reference genome, is GRCh37.p13 with reference to genome version；

3) cuffquant quantifies the expression quantity and normalization output of lncRNA；

4) cuffdiff packet compares control group with the differential expression of disease group lncRNA.

5, result

Sequencing result shows that RP11-320G24.1 expresses significant up-regulation in patients with gastric adenocarcinoma, prompts RP11- 320G24.1 may be applied to the early diagnosis of sdenocarcinoma of stomach as detection target.

The differential expression of embodiment 2QPCR sequence verification RP11-320G24.1 gene

1, the 31 patients with gastric adenocarcinoma cancerous tissue samples and cancer beside organism's sample pair collected according to the collection mode of embodiment 1 RP11-320G24.1 gene differential expression carries out large sample QPCR verifying.

2, RNA is extracted

The extraction of total serum IgE is carried out using the total RNA from animal tissues extracts kit (catalog number (Cat.No.) DP431) of Tiangeng, specifically Step is referring to embodiment 1.

3、QPCR

According to the gene order design primer of RP11-320G24.1 and GADPH, primer sequence is as follows:

RP11-320G24.1:

Forward primer: 5 '-GTATAATCTGCCACTTCT-3 ' (SEQ ID NO.1)

Reverse primer: 5 '-GTCTTCCACATCATTCTA-3 ' (SEQ ID NO.2)

GAPDH:

Forward primer: 5 '-AATCCCATCACCATCTTCCAG-3 ' (SEQ ID NO.3)

Reverse primer: 5 '-GAGCCCCAGCCTTCTCCAT-3 ' (SEQ ID NO.4)

Use Quant one-step method reverse transcription-fluorescence quantitative kit (SYBR Green) kit (catalogue of Tiangeng company Number: PCR reaction NG105) is carried out, reaction system and reaction condition is as shown in table 1.In Thermal Cycler Real PCR amplification is carried out on Time System amplification instrument, confirms that the amplification curve of Real Time PCR and dissolution are bent after reaction Line, Δ Δ CT method carry out relative quantification.

1 QPCR reaction system and reaction condition of table

4, result

QPCR result is as shown in Figure 1, compared with the control, RP11-320G24.1 expresses up-regulation, difference in gastric adenocarcinoma tissue Consistent with high-flux sequence result with statistical significance (P < 0.05), prompting can be by the level of detection RP11-320G24.1 Judge whether subject suffers from sdenocarcinoma of stomach, when the level of RP11-320G24.1 dramatically increases, subject with sdenocarcinoma of stomach or In the presence of the risk for suffering from sdenocarcinoma of stomach, can be designed by the relationship between RP11-320G24.1 and sdenocarcinoma of stomach reduces RP11- The RNA interfering of 320G24.1 level to treat sdenocarcinoma of stomach, while can the relationship based on RP11-320G24.1 and sdenocarcinoma of stomach, The computation model of building prediction sdenocarcinoma of stomach.

The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.

Sequence table

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Claims (10)

1. detecting application of the reagent of long-chain non-coding RNA RP11-320G24.1 in the product for preparing diagnosis of gastric cancer.

2. application according to claim 1, which is characterized in that RP11-320G24.1 expresses up-regulation in patients with gastric cancer.

3. application according to claim 1 or 2, which is characterized in that the product includes: miscellaneous by sequencing technologies, nucleic acid The expression of friendship technology, the method for nucleic acid amplification technologies detection RP11-320G24.1 gene.

4. application according to claim 3, which is characterized in that the nucleic acid amplification technologies be selected from polymerase chain reaction, Reverse transcriptase polymerase chain reaction, the amplification of transcriptive intermediate, ligase chain reaction, strand displacement amplification and based on nucleic acid sequence Amplification.

5. the product of RP11-320G24.1 expression in a kind of vitro detection sample, it is characterised in that the product includes core Piece, kit or nucleic acid film item.

6. product according to claim 5, which is characterized in that it is characterized in that, the chip, kit or nucleic acid film item Specific primer pair or probe including being directed to RP11-320G24.1.

7. product according to claim 6, which is characterized in that the specific primer to for SYBR Green, The detection of Taqman probe, molecular beacon, double cross probe, combined probe.

8. product according to claim 6, which is characterized in that the sequence of the specific primer pair such as SEQ ID NO.1 With shown in SEQ ID NO.2.

9. application of the described in any item products of claim 5-8 in the tool for preparing diagnosis of gastric cancer.

10.RP11-320G24.1 the application in the computation model of building prediction gastric cancer.