CN110257516A - For developing molecular marker and the application of diagnosing gastric cancer product - Google Patents

Abstract

The invention discloses for developing diagnosing gastric cancer product molecular marker and application, the invention discloses molecular marker RP11-199F11.2 to express up-regulation in patients with gastric adenocarcinoma, and the invention discloses application of the RP11-199F11.2 in the product of preparation diagnosis sdenocarcinoma of stomach.

Description

For developing molecular marker and the application of diagnosing gastric cancer product

Technical field

The invention belongs to biomedicine field, it is related to the molecular marker and application for developing diagnosing gastric cancer product.

Background technique

Gastric cancer (Gastric Cancer, GC) is a kind of disease of high mortality, occupies the of global cancer related mortality Two.2011, there are more than 40 ten thousand patients to be diagnosed as gastric cancer in China, and because of about 300,000 people (Di of the patient of mortality of gastric carcinoma Gesualdo F,Capaccioli S,Lulli M.A pathophysiological view of thelong non- codingRNA world.Oncotarget.2014；5:10976-10996.).Environmental factor and life style are to cause this The crucial cause of disease of phenomenon.Although the therapeutic scheme of patients with gastric cancer, still in continuously improving, operation and systemic chemotherapy are main at present Treatment method (Kanat O, the O'Neil BH.Metastatic gastric cancer treatment:a little wanted slowbut worthy progress.Med Oncol.2013；30:464.).However, to the Mechanism Study of gastric cancer occurrence and development It is continued for carrying out, but definite molecular mechanism is still fuzzy.The change and gastric cancer susceptibility of some genes can be specified at present It is related, such as oncogene, factor (Jemal A, Bray F, Center MM, the Ferlay J, Ward of tumor suppressor gene and growth E,Forman D.Global cancerstatistics.CA Cancer J Clin.2011；61:69-90.).Therefore, in order to Gastric cancer early diagnosis is improved, improves prognosis, there is an urgent need to find specific molecular marker in diagnosis.

Long-chain non-coding RNA (long non-coding RNA, 1ncRNA) is a kind of RNA for being usually more than 200nt, is not had There is open reading frame (Rinn JL, Chang HY.Genome regulation by long noncodingRNAs.Annu Rev Biochem.2012；81:145-166.).It is classified as 1ncRNA between gene according to its positioning and transcriptional orientation, 1ncRNA 1ncRNAs (intronic lncRNAs) in (intergenic lncRNAs) and gene (Di Gesualdo F, Capaccioli S,Lulli M.A pathophysiologicalview of the long non-coding RNA world.Oncotarget.2014；5:10976-10996.doi:10.18632/oncotarget.2770.).In nucleus In, the genetic transcription of 1ncRNA major regulatory and mRNA splicing, while they influence stability in ribonucleic acid cytoplasm and MicroRNA (miRNA) activity.Gastric cancer correlation 1ncRNAs is widely studied.LncRNAs participates in multiple tumor signal accesses, such as Notch, mTOR, NF-Kb, Wnt etc..LncRNA participates in cell cycle regulation, proliferation, apoptosis, invasion and transfer.In addition, more next It is more research shows that the unconventionality expression of 1ncRNAs has clinical meaning to the diagnosis of cancer.It studies relevant to sdenocarcinoma of stomach LncRNA for realizing that the early diagnosis of sdenocarcinoma of stomach has great importance, while also becoming the hot spot studied at present.

Summary of the invention

In order to make up for the deficiencies of the prior art, the present invention passes through high throughput sequencing technologies combination and the side of bioinformatics Method screens the lncRNA molecular marker that differential expression is presented in sdenocarcinoma of stomach, by the expression of detection molecules marker, It is compared with reference level, to judge whether subject suffers from sdenocarcinoma of stomach or with the presence or absence of the risk for suffering from sdenocarcinoma of stomach.Together When the molecular marker can be used as the specific molecular target of sdenocarcinoma of stomach, the accurate treatment applied to sdenocarcinoma of stomach.

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

The present invention provides application of the reagent of detection RP11-199F11.2 in the product of preparation diagnosis sdenocarcinoma of stomach.

Further, when timing in RP11-199F11.2 expression, subject is with sdenocarcinoma of stomach or exists with sdenocarcinoma of stomach Risk.

Further, the product includes detecting sample by Nucleic acid sequencing techniques, nucleic acid hybridization technique, nucleic acid amplification technologies The reagent of the expression of middle RP11-199F11.2.

The exemplary, non-limitative example of Nucleic acid sequencing techniques includes but is not limited to chain terminator (Sanger) sequencing and dye Expect terminator sequencing.Those skilled in the art it will be recognized that due to RNA in cell less stable and in an experiment It is more vulnerable to nuclease attack, therefore usually by RNA reverse transcription at DNA before sequencing.

The another exemplary non-limiting example of Nucleic acid sequencing techniques includes that (deep sequencing/high pass measures for next-generation sequencing Sequence), high throughput sequencing technologies are a kind of sequencing technologies in synthesis based on unimolecule cluster, based on proprietary reversible termination chemistry Reaction principle.The random fragment of the DNA of genome is attached to optically transparent glass surface when sequencing, these DNA fragmentations warp After crossing extension and bridge amplification, hundreds of millions of clusters is formed in glass surface, each cluster is the list with thousands of parts of same templates Then molecular cluster utilizes four kinds of special deoxyribonucleotides with fluorophor, skill is sequenced in synthesis by reversible Template DNA to be measured is sequenced in art.

The exemplary, non-limitative example of nucleic acid hybridization technique 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 a part or slice (original position) are the spy in entire tissue (full organization embedding ISH) if tissue is sufficiently small The hybridization of anisotropic DNA or RNA sequence.DNA ISH can be used for determining the structure of chromosome.RNA ISH is for measuring and positioning group Knit the mRNA and other transcripts (for example, ncRNA) in slice or full organization embedding.Usually to sample cell and tissue at Reason increases the entrance of probe with fixation in situ target transcript.Probe hybridizes with target sequence at high temperature, then by extra spy Needle is washed off.Use autoradiograph, fluorescence microscopy or immunohistochemistry respectively, in tissue with radiation, fluorescence or antigen The probe of the kilobase marker of label is positioned and is quantified.Two or more can also be used by radioactivity by ISH or other are non- The probe of radioactive label substance markers, to detect two or more transcripts simultaneously.

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.

The present invention can before detection or simultaneously expand nucleic acid (for example, ncRNA).The example of nucleic acid amplification technologies Property non-limiting example includes but is not limited to: polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), Amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA) and the amplification based on nucleic acid sequence of transcriptive intermediate (NASBA).Those skilled in the art are it will be recognized that certain amplification techniques (for example, PCR) are needed RNA before amplification Reverse transcription is at DNA (for example, RT-PCR), and other amplification techniques then direct cloning RNA (for example, TMA and NASBA).

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.

Further, the reagent is selected from: the probe of specific recognition RP11-199F11.2；Or

The primer of specific amplification RP11-199F11.2；Or

The chip of specificity analysis RP11-199F11.2.

Further, the primer sequence of the specific amplification RP11-199F11.2 is as shown in NO.1~2 SEQ ID.

The present invention provides a kind of product of vitro detection RP11-199F11.2 expression, the product include chip, Kit.

Further, the chip includes: solid phase carrier and specific recognition RP11-199F11.2 attached to it Probe.

As non-limiting embodiment, the various common used materials in genetic chip field are can be used in the solid phase carrier, such as But it is not limited to nylon membrane, the slide or silicon wafer, unmodified slide, plastic sheet modified through active group (such as aldehyde radical, amino) Deng.

The conventional manufacturing method of biochip known in the art can be used in the preparation of chip of the present invention.For example, if solid Phase carrier is gone here and there using modification slide or silicon wafer, 5 ' ends of probe containing amido modified poly- dT, can be by oligonucleotide probe It is configured to solution, then uses point sample instrument that its point on modification slide or silicon wafer, is arranged in scheduled sequence or array, then It is fixed by standing overnight, so that it may obtain lncRNA chip of the invention.

Term " probe " is intended to cover under conditions of promotion hybridizes and the target sequence specificity in nucleic acid or its complement Hybridization, to allow to detect the nucleic acid oligomer or aptamer of target sequence or the nucleic acid of its amplification.Detection can be directly (i.e. by The probe directly hybridized with the sequence of target or amplification generates) or indirectly (i.e. by the sequence with linking probe and target or amplification Middle element structure hybridization probe generate)." target " of probe be often referred to amplification nucleic acid sequence in at least partly The sequence that probe sequence passes through standard hydrogen bond or " base pairing " specific hybrid.The sequence of " substantially complementary " allows probe sequence Column hybridize with target sequence stabilization, even if two sequences are not fully complementary.Probe can be labeled or not mark.Probe can pass through The molecular cloning production of specific DNA sequence dna, can also be synthesized.Those skilled in the art in the invention can easily determine can be A variety of primer and probes of design and use under background of the invention.

" hybridization " or " nucleic acid hybridization " or " hybridization ", which are often referred to two, has complementary base sequence, under proper condition by shape At the hybridization of the single stranded nucleic acid molecule of thermodynamically stable duplex structure.Term " hybridization " as used herein can refer to stringent Or the hybridization under nonstringent condition.The setting of condition within the technical scope of those skilled in the art, can be said according in this field Bright experimental program determines.

Further, the kit includes: the primer of specific amplification RP11-199F11.2, specific recognition RP11- The probe of the probe of 199F11.2 or the chip of specificity analysis RP11-199F11.2.

Term " primer " indicates oligonucleotides, and either naturally occurring in the restrictive digestion content of purifying or synthesis produces It is raw, under conditions of being placed in the induction primer extension product complementary with nucleic acid chains and synthesizing, i.e., there are nucleotide and inducers such as Archaeal dna polymerase and it can be as synthesis starting point when under suitable temperature and pH.Primer can be single-stranded or double-stranded and necessary sufficient It is enough long to cause synthesis expected extension products in the presence of inducer.The definite length of primer depends on several factors, wherein Including temperature, Primer Source and application method.For example, for diagnostic application, dependent on the complexity of target sequence, antisense oligonucleotide primer Object usually contains 15-25 or more, although it can contain more Oligonucleotide.It participates in determining primer suitable length Factor is readily apparent that those skilled in the art.

Further, the kit further includes one or more substances selected from the group below: container, operation instructions, the positive Reference material, negative control object, buffer, auxiliary agent or solvent.

The present invention provides the products of vitro detection RP11-199F11.2 expression in the tool for preparing diagnosis sdenocarcinoma of stomach In application.

The present invention provides application of the RP11-199F11.2 in the pharmaceutical composition of preparation treatment sdenocarcinoma of stomach, the medicines Compositions include the inhibitor of RP11-199F11.2.The inhibitor is that can reduce horizontal any of RP11-199F11.2 Reagent.As non-limiting embodiment, comprising: shRNA (children purpura nephritis), siRNA (siRNA), dsRNA, small RNA, antisense nucleic acid, or can express or be formed the building of the shRNA, siRNA, dsRNA, Microrna, antisense nucleic acid Object.

In the present invention, RP11-199F11.2 gene is located on No. 17 chromosomes, including RP11-199F11.2 gene and Its homologue, mutation and isoform.The term covers overall length, unprocessed RP11-199F11.2, and is originated from cell Any type of RP11-199F11.2 of processing.The term covers natural generation variant (such as the montage of RP11-199F11.2 Variant or allelic variant).A kind of sequence of representative RP11-199F11.2 is as shown in ENST00000571370.1.

Term " expression of difference molecular marker " and " differential expression " are used interchangeably, and are referred to relative to it in normal subjects Expression, or relative to its expression in patient differently reply particular treatment or with different prognosis, table Up to the molecular marker for being activated as higher or lower level in the object with specified disease.The term further includes its expression The molecular marker of higher or lower level is activated as in the different stages of identical disease.It is also to be understood that difference table It can be activated or be suppressed in nucleic acid level or protein level up to molecular marker, or alternative splicing can be subjected to generate not Same polypeptide product.This species diversity can be by including mRNA level in-site, Microrna level, lncRNA level, anti-sense transcript level Or other a variety of changes divided of protein surface expression, secretion or polypeptide confirm.Difference molecular marker is expressed The comparison of expression between two or more genes or between its gene product；Or between two or more genes or its base Because of the comparison of the ratio of the expression between product；Or the comparison of the product of even mutually isogenic two different processing, just It is different between normal object and diseased subjects；Or it is different in the different phase of same disease.Differential expression includes for example Between normal cell and diseased cells or between the cell for undergoing different illness events or disease stage, in molecular marker Difference qualitatively and quantitatively in object in transient expression mode or cell expression pattern.

When molecular marker indicated in individual abnormal process, disease or other illnesss or as abnormal process, disease or When the mark of other illnesss, the molecular marker in individual indicate normal procedure, without disease or other illnesss or as just Chang Jincheng, mark without disease or other illnesss molecular marker expression or value compare, be described generally as It is expression or low expression." up-regulation ", " up-regulation ", " overexpression ", " overexpression " are used interchangeably, refer to be greater than health or Point in the biological sample of the value or level (or range of value or level) of the molecular marker typically detected in normal individual The value or level of sub- marker.The term also can refer to be greater than in the detectable molecular marker of the different phase of specified disease The value or level of molecular marker in the biological sample of value or horizontal (or range of value or level).

" downward ", " downward ", " low expression ", " low expression " are used interchangeably, and are referred to and are less than in health or normal individual In molecular marker in the biological sample of the value of molecular marker or level (or range of value or level) that typically detects Value or level.The term also can refer to be less than the value or level in the detectable molecular marker of the different phase of specified disease The value or level of molecular marker in the biological sample of (or range of value or level).

In the present invention, it can be provided in kit for reagent described herein, tool and/or specification.For example, Kit may include for determining the reagent suitably treated, tool and specification about cancer patient.The kit can wrap The reagent for collecting tissue sample from patient is included, such as is collected by biopsy, and the reagent for handling the tissue.Reagent Box may also include one or more reagents for carrying out molecular marker expression analysis, for example, for carry out RT-PCR, qPCR, The reagent of the expression of molecular marker in the sample to determine patient such as RNA trace.For example, can be wrapped in the kit The primer for carrying out RT-PCR is included, for carrying out the probe of rna blot analysis.It may also include the buffering appropriate for measurement Liquid.Detection reagent needed for may also include any one of these measurements.

The kit characterized herein, which may also include, describes how to carry out the measurement for measuring molecular marker expression Instruction card.Instruction card may also include the explanation how determined with reference to group, include how to determine with reference to molecular marker in group Expression and how to gather expression data with establish for test patient compared with reference.Instruction card may also include use In measurement test patient in molecular marker expression and for by the expression with refer to group in expression compare from And determine the explanation of the chemotherapy appropriate for subject.

The information material for including in kit can be with approach described herein and/or for side described herein The relevant description of the purposes of the reagent of method, guidance, sale or other materials.For example, the information material of kit can Comprising contact details, for example, physical address, e-mail address, website or telephone number, wherein the user of kit can be obtained The bulk information in relation to carrying out gene expression analysis and parsing result is obtained, it is especially positive when being applied to have particular therapeutic agent When the mankind of property response.

Detailed description of the invention

Fig. 1 is the expression figure using QPCR detection RP11-199F11.2 gene in gastric adenocarcinoma tissue.

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: utilizing DNA fragmentation present in DNase I digestion Total RNA sample, magnetic bead Purification and recovery 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 5 ' adapter of cDNA: 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 StepOnePlusReal-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 is shown, compared with cancer beside organism, RP11-199F11.2 expresses significant up-regulation in patients with gastric adenocarcinoma, Prompt RP11-199F11.2 that may be applied to the early diagnosis of sdenocarcinoma of stomach as detection target.

The differential expression of 2 QPCR sequence verification RP11-199F11.2 gene of embodiment

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 RP11-199F11.2 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 RP11-199F11.2 and GADPH, primer sequence is as follows:

RP11-199F11.2:

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

Reverse primer: 5 '-CAAATGGGAAACAACTCAAA-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 below.In Thermal CyclerReal 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.

The configuration of 50 μ l reaction systems

Following reagent is added into EP pipe and mixes: 2 × Quant One Step RT-qPCR Mix (SYBR) 25 μ l, 2.5 μ l of Hotmaster Taq Polymerase 2.5U/ μ l, 0.4 μ l of Quant RTase, it is positive it is (anti-) to 0.2 μM of primer, it is total RNA 50ng adds nuclease free water to 50 μ l.

Reaction condition

50 DEG C of 30min, 95 DEG C of 2min；

(94 DEG C of 20s, 55 DEG C of 20s, 68 DEG C of 20s) × 40

4, result

QPCR result is as shown in Figure 1, compared with the control, RP11-199F11.2 expresses up-regulation in gastric adenocarcinoma tissue, up-regulation About 2.9 times, difference has statistical significance (P < 0.05), consistent with high-flux sequence result, and prompt can be by detecting RP11- The level of 199F11.2 judges whether subject suffers from sdenocarcinoma of stomach, compared with normal control, when the level of RP11-199F11.2 is aobvious Write when increasing, subject is with sdenocarcinoma of stomach or there is the risk for suffering from sdenocarcinoma of stomach, by RP11-199F11.2 and sdenocarcinoma of stomach it Between relationship can design the RNA interfering for reducing RP11-199F11.2 level to treat 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.

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Claims (10)

1. detecting application of the reagent of RP11-199F11.2 in the product of preparation diagnosis sdenocarcinoma of stomach.

2. application according to claim 1, which is characterized in that when timing, subject suffer from RP11-199F11.2 expression There is the risk for suffering from sdenocarcinoma of stomach in sdenocarcinoma of stomach.

3. application according to claim 1, which is characterized in that the product includes hybridizing skill by sequencing technologies, nucleic acid The reagent of the expression of RP11-199F11.2 gene in art, nucleic acid amplification technologies detection sample.

4. application according to claim 3, which is characterized in that the reagent is selected from:

The probe of specific recognition RP11-199F11.2；Or

The primer of specific amplification RP11-199F11.2；Or

The chip of specificity analysis RP11-199F11.2.

5. application according to claim 4, which is characterized in that the primer sequence of the specific amplification RP11-199F11.2 Column are as shown in NO.1~2 SEQ ID.

6. a kind of product of vitro detection RP11-199F11.2 expression, which is characterized in that the product includes chip, examination Agent box.

7. product according to claim 6, which is characterized in that the chip includes: solid phase carrier and attached to it Specific recognition RP11-199F11.2 probe.

8. product according to claim 7, which is characterized in that the kit includes: specific amplification RP11- The primer of 199F11.2, the probe or specificity of the probe of specific recognition RP11-199F11.2 analyze RP11-199F11.2's Chip.

9. product according to claim 8, which is characterized in that the kit further includes selected from the group below one or more Substance: container, operation instructions, positive control, negative control object, buffer, auxiliary agent or solvent.

10. application of the described in any item products of claim 5-9 in the tool of preparation diagnosis sdenocarcinoma of stomach.