CN104877998A - Long noncoding RNA (LncRNA), and primer pair and kit for detecting expression level of long noncoding RNA in cells and tissues - Google Patents
Long noncoding RNA (LncRNA), and primer pair and kit for detecting expression level of long noncoding RNA in cells and tissues Download PDFInfo
- Publication number
- CN104877998A CN104877998A CN201510242287.3A CN201510242287A CN104877998A CN 104877998 A CN104877998 A CN 104877998A CN 201510242287 A CN201510242287 A CN 201510242287A CN 104877998 A CN104877998 A CN 104877998A
- Authority
- CN
- China
- Prior art keywords
- cancer
- primer
- lncrna
- stomach
- primer pair
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a new long noncoding RNA (LncRNA) Lnc21q22.11 sequence, and a primer pair and kit for detecting expression level of the long noncoding RNA in cells and tissues. The full length of the LncRNA Lnc21q22.11 is identified to be 1202bp for the first time, the specific primers are utilized for the first time to detect the expression level of the LncRNA Lnc21q22.11 in stomach cancer cells, 35 pairs of stomach cancer line and para-carcinoma tissues, colorectal cancer cell lines and 10 pairs of colorectal cancer and para-carcinoma tissues, and thus, the kit is creative. The detection of the expression of the LncRNA Lnc21q22.11 in the stomach cancer tissues by using the kit and specific primers can be used as an effective means for stomach cancer and colorectal cancer diagnosis, curative effect observation, prognosis judgment, focus residue and relapse detection and the like. The kit is simple to operate, has the advantages of high stability and high sensitivity, and has far-reaching clinical meanings and popularization performance.
Description
Technical field
The present invention relates to a kind of new long-chain non-coding RNA and for detecting the primer of this long-chain non-coding RNA expression level in cell and tissue and test kit.
Background technology
According to global cancer epidemiology statistic data (GLOBOCAN2012) in 2012, the nearly 1,000,000 cancer of the stomach new cases (952000,6.8%) in 2012 were the 5th common cancer in the whole world; Cancer of the stomach is the 3rd (723000,8.8%) in the lethal cancer in the whole world.Occur in developing country more than the gastric cancer cases of 70%, 50% cancer of the stomach of world wide sum occurs in East Asia Region, mainly in China.Cancer of the stomach is malignant tumour (Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray, the F. of sickness rate second lethality rate the 3rd in China
GLOBOCAN 2012v1.0, Cancer Incidence and Mortality Worldwide:IARC CancerBase No.11 [Internet] .Lyon, France:International Agency forResearch on Cancer; 2013.Available from:http: //globocan.iarc.fr, accessedon day/month/year.) high incidence of cancer of the stomach and high lethality rate serious threat the health of the mankind, a lot of patients with gastric cancer is just in progressive stage when making a definite diagnosis, and current excision is the means of uniquely effecting a radical cure cancer of the stomach.Early screening and diagnosis become the core means of cancer of the stomach prediction, and simultaneously gastroscopy merges the diagnosis that biopsy contributes to early gastric cancer type, but make it cannot become the extensive examination means of cancer of the stomach due to gastroscopic costliness.The tumor markers analytical procedure of current applied examination cancer of the stomach is simple and quick, but its sensitivity and low (the Ming-Ming Tsai of specificity, Chia-Siu Wang, etc, Potential prognostic, diagnostic and therapeuticmarkers for human gastric cancer, World J Gastroenterol 2014October 14; 20 (38): 13791-13803).Therefore find that the effective ways tool of the early diagnosis and therapy of cancer of the stomach is of great significance.
Colorectal cancer is the 3rd common cancer (746,000 example, accounts for 10.0% of sum) in the world in the male sex, it is the second common cancer (614 in women, 000 example, accounts for 9.2% of sum), the case of about 55% occurs in developed country.The mortality ratio of colorectal cancer accounts for 8.5% of total cancer mortality, colorectal cancer causes death to mostly occur in low developed area, and poor prognosis (FerlayJ, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray, F.GLOBOCAN 2012v1.0, Cancer Incidenceand Mortality Worldwide:IARC CancerBase No.11 [Internet] .Lyon, France:International Agency for Research on Cancer; 2013.Available from:http: //globocan.iarc.fr, accessed on day/month/year.).In the last few years due to the development of the universal of tumor screening and diagnostic means, new cases and the death of colorectal cancer decline all to some extent, but still have an appointment every year in the whole world, 783000 new colorectal cancer cases are diagnosed, and local recurrence and distant metastasis can occur only about half of colorectal cancer patients.The main treatment means of colorectal cancer is chemotherapy, and Advanced colorectal cancer is considered to more tolerate chemotherapy (MyoungSookKim, JunaLeeandDavidSidransky.DNAmethylationmarkersincolorect alcancer.CancerMetastasisRev (2010) 29:181 – 206) for a long time.Therefore the discovery of colorectal cancer early diagnosis marker and the discovery of effective treatment have great importance.
Sequence only less than 2% in human genome can coded protein, but the human genome DNA of 70% ~ 90% is transcribed, its transcription product is considered to " dark matter " or " rubbish " in the past, it is found that these transcription products can participate in the vital movement such as division, differentiation of cell in recent years, have important biological function.Non-coding RNA comprises many types such as miRNA, piRNA and long-chain non-coding RNA, and wherein long-chain non-coding RNA receives the concern of people gradually.Long-chain non-coding RNA and length are greater than 200nt and lack the RNA of open reading frame, most of long-chain non-coding RNA has polyA tail (Kung, J.T., D.Colognori, and J.T.Lee, Long noncoding RNAs:past, present, and future.Genetics.193 (3): p.651-69, 2013.Esteller, M., Non-coding RNAs in human disease.Nat Rev Genet.12 (12): p.861-74, 2011.Mercer, T.R., M.E.Dinger, and J.S.Mattick, Long non-coding RNAs:insights into functions.Nat Rev Genet.10 (3): p.155-9, 2009.).LncRNA can transcribing, to translate and the expression of post-transcriptional level to gene regulates and controls (Gutschner, T.and S.Diederichs, The hallmarks ofcancer:a long non-coding RNA point of view.RNA Biol.9 (6): p.703-19,2012.).
Along with the development of high throughput sequencing technologies and the maturation of RNA chip technology, increasing lncRNA is found, but the function of most of lncRNA indefinite.There is research display, lncRNA plays a significant role (Maruyama in the physiology such as cell proliferation, cell cycle, apoptosis, invasion and attack migration and pathologic process, R.and H.Suzuki, Long noncoding RNAinvolvement in cancer.BMB Rep.45 (11): p.604-11,2012.).The lncRNA of the research comparative maturity relevant to tumour mainly contains MALAT1, PANDA, PCAT-1 etc.MALAT1 (Metastasis-Associated Lung Adenocarcinoma Transcript 1) at Several Kinds of Malignancy as liver cancer, equal up-regulated in endometrial stromal sarcoma and lung cancer, in the lung cancer of transfer, the expression amount of MALAT1 is three times of non-diverting lung cancer, the independent prognostic index (Ji assessing early stage survival time of lung adenocarcinoma, P., et al., MALAT-1, a novelnoncoding RNA, and thymosin beta4predict metastasis and survival inearly-stage non-small cell lung cancer.Oncogene.22 (39): p.8031-41, 2003.Yamada, K., et al., Phenotypic characterization of endometrial stromalsarcoma of the uterus.Cancer Sci.97 (2): p.106-12, 2006.Lin, R., et al., Alarge noncoding RNA is a marker for murine hepatocellular carcinomas anda spectrum of human carcinomas.Oncogene.26 (6): p.851-8, 2007.).PANDA is high expression level in human breast cancer, the mark (Hung of mammary cancer chemotherapy resistance, T., et al., Extensive and coordinated transcription of noncoding RNAs withincell-cycle promoters.Nat Genet.43 (7): p.621-9,2011).PCAT1 (prostatecancer associated transcript-1) is by the lncRNA of specificity overexpression in prostate cancer that high-throughput transcript sequencing technologies obtains in cases for prostate cancer, another lncRNAPCA3 (The prostate cancer antigen-3gene) is used for early detection and prognosis evaluation (the Prostatecancer:Biomarkers PCA3and TMPRSS2-ERG:better together.Nat RevUrol of prostate cancer by U.S. food and drug administration (FDA) in approval in 2012, 2014.Porpiglia, F., et al., The roles of multiparametric MRI, PCA3, and PHI:which is the best predictor of prostate cancer after anegative biopsy? Results of a prospective study.J Urol, 2014.Schroder, F.H., et al., Prostate cancer antigen 3:diagnostic outcomes inmen presenting with urinary prostate cancer antigen 3scores>/=100.Urology.83 (3): p.613-6, 2014.).But the research that relevant lncRNA occurs to develop to cancer of the stomach is still few, the mechanism of action of lncRNA in cancer of the stomach and clinical value still need to be studied.
Summary of the invention
Base sequence involved in the present invention long-chain non-coding RNA as shown in SEQ ID NO:1 (Long noncoding RNA, LncRNA) (be denoted as Lnc21q22.11 below) and be by normal gastric mucosa and 3 strain gastric carcinoma cell lines (AGS, SGC7901, BGC823) total serum IgE that extracts carried out the order-checking of high-throughput transcript and obtained the lncRNA reduced at expression in gastric carcinoma of Late Cambrian new in the lncRNA of differential expression by bioinformatic analysis.The full length sequence being obtained Lnc21q22.11 by 3 ' RACE (rapid-amplification of cDNA ends) and 5 ' RACE is 1202bp, is positioned at the mankind's No. 21 chromosome long arm.Preliminary result prompting cell Lnc21q22.11 may be cancer suppressor gene new in cancer of the stomach, may be a brand-new tumor molecular marker in stomach organization to the detection of Lnc21q22.11 expression amount.On this basis, the present inventor applies semiquantitive RT-PCR and detects, with the change of the expression amount of clear and definite Lnc21q22.11 in cancer of the stomach a large amount of stomach organization sample and cancer beside organism's sample.
First passage 5 ' RACE and 3 ' RACE technical evaluation Lnc21q22.11 total length 1202bp of the present invention, full length sequence as shown in SEQ ID NO:1, that is:
GCTCAACGGGTTGACGGCGCCCGACGGCCAGGTCCAGCAGGGGCGCCCCGCAGGTGGGGCCTGGCGGCCTTCACCTTCCGGATCCCCGACCCGGCGCGACCGGGAGCCGGCAGACTTTTGTCTAGGAGGGAAAACCCGAGCGCGGGGCCGGCCGCGACATCGCAGCATCCCAAAGAGCTTTTGGAATTTGGGTCACTCCCTTCTACCCCGACGTCACACACTTACTGCTCCCGGCAGCGGAGGCTCCAGCGCCTGGCCGCGCACAAACCACGACTTCTACCGTCCTGCCGGGGAAAACTACAGGTCCCGAAATGCACCGCTACGTGCTCAGGCGCAGTGGGCCGGTGCCGCCGACGAGAGTGCACTACTCCGTGCGCAGGCGCAGTGGGCCCGGGCAGAAGACCTGGGGCGCGCTGCTCACTGCGCAGGCGCAGTGAGCCCAGGCGGGCAGCCCTGGCCAGCAGCTCTTGTCCCTGTGGCTGGAGGCTGAAGTCCACGTAGGCCCCGGCGGGGAGCCGCTGGGGTGTAGGCCGGGTGCCTTTGTCCAAGCCTGGCGCCACTGCCCAGACACTTTACTTCAAATTTGGCTTTATTTTCATCATTGATATTGATTAAAATGACATTCACTTGCCCGGAAAGTAAAAGAGGTCTTCAGCACAAAATATTGCTCCAACAAGCAAGCATAAATCACTGAGATTGAAATGTCATCTATCTTTATTGGCCAACCACATAATCTCCATGAAAATTCTCAACAGAGAGCAAGGAACTCACTTTCTTCTAAGCAAAGACAATTTGGATTTGCCTCGTGGCACTGGCATCTCTTCATGTCTGGACGTCAGATGTTAGCTTGGTGCCTTTCTCCCTTATCAGGGGGACATTTTGAACCCAGCCGCACCAATGTTCTCATGGATGGTGGCCTTCGATTCTTCTAGATTCAAGATTTGGCTCTTGTACTTTTCCTGCCAGTCCTCTACAATGTACTGGTGGTAGGGGTCATTGGAGTGTTCCCGTCTCTTGGATTTCACAGTGCTCACCAGGATGGCCACGATGATGAAAGAGAACATTCCAATCATCACCATGAGGTACAGGATGACATAGTAGAAGTTCTCAGCATCAACTTTGGCTTGGAGGGCCTCTTGCTCAGCTGTTGTGTTCTGGCGCCAATTGTCCATATAAGTAATAAAAATCCTTCGGAAGACGTC。
The present invention also provides a kind of specificity Semiquatitative RT-PCR assay primer pair of the long-chain non-coding RNA Lnc21q22.11 expression level detected in stomach cancer cell and tissue, the upstream primer nucleotide sequence of described primer pair is as shown in SEQ ID NO:2, that is: 5 '-AATTTGGGTCACTCCCTTCTAC-3 ', downstream primer nucleotide sequence as shown in SEQID NO:3, that is: 5 '-GTGAGTTCCTTGCTCTCTGTTG-3 '.
The present invention also provides a kind of test kit for detecting stomach cancer cell and tissue and colorectal cancer cell and tissue, it is characterized in that: this test kit contains above-mentioned primer pair.
Further, test kit of the present invention can also contain the Semiquatitative RT-PCR assay primer of internal reference GAPDH, the Semiquatitative RT-PCR assay primer of this internal reference GAPDH can be the conventional primer in this area, such as its upstream primer can nucleotide sequence as shown in SEQ ID NO:4, that is: 5 '-GACCACAGTCCATGCCATCAC-3 ', downstream primer nucleotide sequence can as shown in SEQ ID NO:5, that is: 5 '-GTCCACCACCCTGTTGCTGTA-3 '.
The cDNA that after this test kit can also extract total serum IgE containing long-chain non-coding RNA high-expression cell line BGC823, reverse transcription also dilutes, as the positive template of the feasibility of the above-mentioned primer pair of detection.
Further, test kit of the present invention can also contain the following ingredients needed for RT-PCR reaction: 5 × PCR buffer, 10mM dNTPs, Hot start Taq enzyme, ddH
2o.
The present invention also provides a kind of RT-PCR reaction system for detecting cancer of the stomach and colorectal cancer, and in cumulative volume 25ul, it comprises:
(1) template: 2.5ul;
(2) concentration is the above-mentioned primer pair of 10uM and the Semiquatitative RT-PCR assay primer of internal reference GAPDH, wherein, and upstream primer and each 1ul of downstream primer;
(3)5×PCR buffer：5ul；
(4)10mM dNTPs:0.5ul；
(5) Hot start Taq enzyme: 0.25ul;
(6)ddH
2O:14.75ul。
Tool of the present invention has the following advantages: utilize primer pair of the present invention and test kit to detect stomach organization and cancer beside organism, in stomach organization, Lnc21q22.11 expresses reduction by 80% (28/35) compared with cancer beside organism, show this lncRNA low expression in cancer of the stomach, be cancer suppressor gene potential in cancer of the stomach, can be used as the new molecular marker of cancer of the stomach; Utilize primer pair of the present invention and test kit to detect Colorectal Carcinoma and cancer beside organism simultaneously, in Colorectal Carcinoma, Lnc21q22.11 expresses reduction by 70% (7/10) compared with cancer beside organism, show this lncRNA low expression in colorectal cancer, be cancer suppressor gene potential in colorectal cancer, can be used as the new molecular marker of colorectal cancer.This test kit selects Lnc21q22.11 as detection mark first, and this lncRNA is the lncRNA expressing decline in stomach organization and colorectal cancer that the present inventor filters out first in cancer of the stomach and colorectal cancer, has initiative.Therefore, apply primer pair provided by the invention and test kit and can be used as to the expression level detecting cancer of the stomach or colorectal cancer Lnc21q22.11 the powerful measure that cancer of the stomach or diagnosis of colorectal carcinoma, observation of curative effect, Index for diagnosis, residual and recurrence detect etc., and, easy and simple to handle, good stability, highly sensitive, there is far-reaching clinical meaning and generalization.
Accompanying drawing explanation
The expression level that Fig. 1 applies above-mentioned Lnc21q22.11 Semiquatitative RT-PCR assay primer pair Lnc21q22.11 in 10 strain gastric carcinoma cell lines detects, and GAPDH is internal reference, ddH
2o is system contrast, whether there is the pollution of PCR primer, as ddH in order to evaluation system
2the result that O detects is negative, then system credible result, body series amplified production size is 587bp.
Fig. 25 to the cancer of the stomach of pairing and cancer beside organism in apply above-mentioned Lnc21q22.11 Semiquatitative RT-PCR assay primer pair and detect the expression level of Lnc21q22.11, GAPDH is internal reference, ddH
2o is system contrast, whether there is the pollution of PCR primer, as ddH in order to evaluation system
2the result that O detects is negative, then system credible result, body series amplified production size is 587bp.
Fig. 3 is in 10 strain colorectal cancer cell systems, and the expression level applying above-mentioned Lnc21q22.11 Semiquatitative RT-PCR assay primer pair Lnc21q22.11 in 10 strain gastric carcinoma cell lines detects, and GAPDH is internal reference, ddH
2o is system contrast, whether there is the pollution of PCR primer, as ddH in order to evaluation system
2the result that O detects is negative, then system credible result, body series amplified production size is 587bp.
Fig. 45 to the colorectal cancer of pairing and cancer beside organism in apply above-mentioned Lnc21q22.11 Semiquatitative RT-PCR assay primer pair and detect the expression level of Lnc21q22.11, GAPDH is internal reference, ddH
2o is system contrast, whether there is the pollution of PCR primer, as ddH in order to evaluation system
2the result that O detects is negative, then system credible result, body series amplified production size is 587bp.
Fig. 5 is by cDNA and the ddH of the gastric carcinoma cell lines BGC823 of Lnc21q22.11 high expression level
2o is mixed in proportion, and apply the expression level that above-mentioned Lnc21q22.11 Semiquatitative RT-PCR assay primer pair detects Lnc21q22.11, amplified band as shown in the figure.BGC823 cell cDNA content is followed successively by 100%, 50%, 5%, 1%, 0.5%, 0%.DdH
2o is system contrast, whether there is the pollution of PCR primer, as ddH in order to evaluation system
2the result that O detects is negative, then system credible result, body series amplified production size is 587bp.
Embodiment
For understanding the present invention in more detail, the present invention is further illustrated by the following examples.
Embodiment one
1, the preparation (extraction of total serum IgE and process of reverse-transcription) of template
The preparation of RNA: obtain cancer beside organism's sample of stomach organization sample and correspondence and cancer beside organism's sample of Colorectal Carcinoma sample and correspondence, respectively get 5 stomach organization sample GC1 ~ GC5 in the present embodiment, 5 cancer beside organism sample GN1 ~ GN5 matched with stomach organization, and respectively get 5 Colorectal Carcinoma sample CRC1 ~ CRC5 in the present embodiment, 5 cancer beside organism sample CN1 ~ CN5 matched with Colorectal Carcinoma.
The method of application TRIZOL extracting carries out the extraction of total serum IgE respectively to above-mentioned sample, UV detector measures absorbancy (A) value and determines its content and purity, 1% agarose gel electrophoresis qualification RNA quality.
CDNA synthesizes: be the first chain cDNA by the reverse transcription of primer immediately in the cancer beside organism of above-mentioned stomach organization and correspondence and in total serum IgE 5ug application SuperscriptIII-reversetranscriptasekit (Invitrogen) of extracting of the cancer beside organism of Colorectal Carcinoma and correspondence, gained cDNA is diluted to 100ul with adding deionized water, gets 2.5ulcDNA wherein for 25ul system Semiquatitative RT-PCR assay.
2, Semiquatitative RT-PCR assay
Carry out the PCR reaction system increased with Lnc21q22.11 Semiquatitative RT-PCR assay primer, in cumulative volume 25ul, comprising:
(1) template (cDNA): 2.5ul;
(2) concentration is the described primer pair of 10uM, wherein, and upstream primer and each 1ul of downstream primer;
Upstream primer nucleotide sequence as shown in SEQ ID NO:2, that is: 5 '-AATTTGGGTCACTCCCTTCTAC-3 ': 1ul,
Downstream primer nucleotide sequence as shown in SEQ ID NO:3, that is: 5 '-GTGAGTTCCTTGCTCTCTGTTG-3 ': 1ul;
(3)5×PCR buffer：5ul；
(4)10mM dNTPs:0.5ul；
(5) Hot start Taq enzyme: 0.25ul;
(6)ddH
2O:14.75ul。
Amplification condition:
Lnc21q22.11 adopts the amplification of Stepdown PCR condition, and condition is as follows:
95℃5min
95 DEG C of 30s → 64 DEG C 30s → 72 DEG C 60s, 3 circulations;
95 DEG C of 30s → 61 DEG C 30s → 72 DEG C 60s, 3 circulations;
95 DEG C of 30s → 58 DEG C 30s → 72 DEG C 60s, 3 circulations;
95 DEG C of 30s → 55 DEG C 30s → 72 DEG C 60s, 26 circulations;
72℃ 5min.
Product size: 587bp
The Semiquatitative RT-PCR assay 25ul system of internal reference GAPDH comprises:
(1) template: 2.5ul;
(2) Semiquatitative RT-PCR assay primer (10pmol/ul):
Upstream primer nucleotide sequence as shown in SEQ ID NO:4, that is: 5 '-GACCACAGTCCATGCCATCAC-3 ': 1ul,
Downstream primer nucleotide sequence as shown in SEQ ID NO:5, that is: 5 '-GTCCACCACCCTGTTGCTGTA-3 ': 1ul;
(3)5×PCR buffer：5ul；
(4)10mM dNTPs:0.5ul；
(5) Hot start Taq enzyme: 0.25ul;
(6)ddH
2O:14.75ul。
Product size: 454bp
Amplification condition is:
Reaction conditions: 95 DEG C of 5min; 95 DEG C of 30s → 63 DEG C 30s → 72 DEG C 45s, 25 circulations; 75 DEG C of 5min, product size: 454bp.
The detection of 3.PCR reaction product
PCR primer carries out 2% agarose gel electrophoresis, and ultraviolet transmission analyser detects and takes a picture.
4. result
The expression amount with Lnc21q22.11 in Colorectal Carcinoma and pairing cancer beside organism in stomach organization and pairing cancer beside organism is judged according to agarose gel electrophoresis PCR primer band power.In 5 pairs of stomach organizations and pairing cancer beside organism, in stomach organization Lnc21q22.11 expression amount lower than pairing cancer beside organism (Fig. 2) 5 pairs of Colorectal Carcinomas and pairing cancer beside organism in, wherein in 3 pairs of colorectal cancers Lnc21q22.11 expression amount lower than pairing cancer beside organism.Wherein GC is stomach organization, and GN is the cancer of the stomach cancer beside organism of pairing, and CRC is Colorectal Carcinoma, and CN is the colorectal cancer cancer beside organism of pairing, and GAPDH is internal reference, ddH
2o is system contrast, whether there is the pollution of PCR primer, as ddH in order to evaluation system
2the result that O detects is negative, then system credible result, body series amplified production size is 587bp.
Embodiment two: clinical samples detects
(1) 35 are got to the stomach organization matched and cancer beside organism, carry out the Semiquatitative RT-PCR assay amplification of Lnc21q22.11, the detection of Template preparation, PCR amplification system and condition, amplified production is all with to implement in one identical, and detected result (expression level is the relative level of stomach organization compared with cancer beside organism of pairing mutually) asks for an interview following table 1:
Table 1
(2) 10 are got to the Colorectal Carcinoma matched and cancer beside organism, carry out the Semiquatitative RT-PCR assay amplification of Lnc21q22.11, the detection of Template preparation, PCR amplification system and condition, amplified production is all with to implement in one identical, and detected result (expression level is the relative level of Colorectal Carcinoma compared with cancer beside organism of pairing mutually) asks for an interview following table 2:
Table 2
Embodiment three: susceptibility is tested
By cDNA and the ddH of the gastric carcinoma cell lines BGC823 of Lnc21q22.11 high expression level
2o is mixed in proportion, and apply the expression level that above-mentioned Lnc21q22.11 Semiquatitative RT-PCR assay primer pair detects Lnc21q22.11, amplified band as shown in Figure 5.BGC823 cell cDNA content is followed successively by 100%, 50%, 5%, 1%, 0.5%, 0%.DdH
2o is system contrast, whether there is the pollution of PCR primer, as ddH in order to evaluation system
2the result that O detects is negative, then system credible result, body series amplified production size is 587bp.
The result display of Fig. 5: with the Auele Specific Primer of the Lnc21q22.11 in the present invention to and reaction system, condition detect cell Lnc21q22.11 and express, its sensitivity can reach 1%, highly sensitive.
Embodiment four: detect the test kit composition that Lnc21q22.11 in cancer of the stomach and colorectal cancer cell expresses
The test kit detecting gastric cancer tumor cell comprises following composition, and wherein the consumption of a Semiquatitative RT-PCR assay is:
1, the RT-PCR primer pair of the concentration Lnc21q22.11 that is 10pmol/ul: upstream primer nucleotide sequence is as shown in SEQ ID NO:2, and downstream primer nucleotide sequence, as shown in SEQ ID NO:3, is respectively 1ul.
2, the RT-PCR primer of the concentration GAPDH that is 10pmol/ul: upstream primer nucleotide sequence is as shown in SEQ ID NO:4, and downstream primer nucleotide sequence, as shown in SEQ ID NO:5, is respectively 1ul.
3, total 2 parts of reaction system, coordinate Lnc21q22.11RT-PCR primer pair and GAPDH RT-PCR primer to try out respectively, every part of reaction system comprises: 5 × PCR buffer, 10mMdNTPs, Hot start Taq enzyme, ddH
2o.
A test kit can comprise the consumption that above-mentioned each composition carries out repeatedly Semiquatitative RT-PCR assay, as 25 times, 50 times, 100 inferior, the Specific amounts of each composition is depending on the circumstances or the needs of the situation determined.
For preventing false positive and the false negative of Semiquatitative RT-PCR assay amplified production, test kit also comprises following several compositions:
(1) positive control pcr template: the cDNA that after the normal gastric mucosa extraction total serum IgE of high expression level Lnc21q22.11, reverse transcription also dilutes, the consumption carrying out a RT-PCR is: 2.5ul.
(2) negative system contrast: ddH
2whether O, exist the pollution of PCR primer, as ddH in order to evaluation system
2the result that O detects is negative, then system credible result.
Claims (7)
1. a long-chain non-coding RNA molecule, its base sequence is as shown in SEQ ID NO:1.
2. a primer pair, it is characterized in that, for detecting the long-chain non-coding RNA expression level as claimed in claim 1 in biological cells and tissues, the upstream primer nucleotide sequence of this primer pair is as shown in SEQ ID NO:2, that is: 5 '-AATTTGGGTCACTCCCTTCTAC-3 ', downstream primer nucleotide sequence as shown in SEQ ID NO:3, that is: 5 '-GTGAGTTCCTTGCTCTCTGTTG-3 '.
3. detect a test kit for cancer of the stomach and colorectal cancer, it is characterized in that: containing primer pair according to claim 2.
4. test kit according to claim 3, it is characterized in that: the Semiquatitative RT-PCR assay primer also containing internal reference GAPDH, its upstream primer nucleotide sequence is as shown in SEQ ID NO:4, that is: 5 '-GACCACAGTCCATGCCATCAC-3 ', downstream primer nucleotide sequence as shown in SEQ ID NO:5, that is: 5 '-GTCCACCACCCTGTTGCTGTA-3 '.
5. the test kit according to claim 3 or 4, it is characterized in that: the cDNA that after also extracting total serum IgE containing long-chain non-coding RNA high-expression cell line BGC823, reverse transcription also dilutes, requires the positive template of the feasibility of the primer pair described in 2 as test right.
6. the test kit according to claim 3 or 4, is characterized in that: also containing the following ingredients needed for RT-PCR reaction: 5 × PCR buffer, 10mM dNTPs, Hot start Taq enzyme, ddH
2o.
7., for detecting a RT-PCR reaction system for cancer of the stomach and colorectal cancer, in cumulative volume 25ul, it comprises:
(1) template: 2.5ul;
(2) the Semiquatitative RT-PCR assay primer of the concentration primer pair according to claim 2 that is 10uM and the internal reference GAPDH described in claim 4, wherein, upstream primer and each 1ul of downstream primer;
(3)5×PCR buffer：5ul；
(4)10mM dNTPs:0.5ul；
(5) Hot start Taq enzyme: 0.25ul;
(6)ddH
2O:14.75ul。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510242287.3A CN104877998B (en) | 2015-05-13 | 2015-05-13 | The primer pair and kit of the long-chain non-coding RNA expression in long-chain non-coding RNA and detection cell and tissue |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510242287.3A CN104877998B (en) | 2015-05-13 | 2015-05-13 | The primer pair and kit of the long-chain non-coding RNA expression in long-chain non-coding RNA and detection cell and tissue |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104877998A true CN104877998A (en) | 2015-09-02 |
| CN104877998B CN104877998B (en) | 2018-03-30 |
Family
ID=53945675
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510242287.3A Active CN104877998B (en) | 2015-05-13 | 2015-05-13 | The primer pair and kit of the long-chain non-coding RNA expression in long-chain non-coding RNA and detection cell and tissue |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104877998B (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106676191A (en) * | 2017-03-03 | 2017-05-17 | 中日友好医院 | Molecular marker for colon adenocarcinoma |
| CN107043823A (en) * | 2017-05-26 | 2017-08-15 | 郴州市第人民医院 | A kind of related tumor markers of colorectal cancer and application |
| CN107119125A (en) * | 2017-05-11 | 2017-09-01 | 青岛大学 | The application of lncRNA D63785 and its chaff interference and apply its product |
| CN108034655A (en) * | 2017-10-26 | 2018-05-15 | 南京医科大学第二附属医院 | A kind of application of the long non-coding RNA and combinations thereof in diagnosis/treatment colorectal cancer |
| CN108753960A (en) * | 2018-04-24 | 2018-11-06 | 郑州大学第附属医院 | A kind of gastric cancer prognosis molecule marker non-coding RNA Lnc-DENR-2 and its application |
| CN108841964A (en) * | 2018-08-08 | 2018-11-20 | 王冬国 | Application of the LOC105370108 in rectal adenocarcinoma diagnosis and treatment |
| CN108866198A (en) * | 2018-08-31 | 2018-11-23 | 北京泱深生物信息技术有限公司 | Application of the marker LOC105376380 in rectal adenocarcinoma diagnosing and treating |
| CN108949991A (en) * | 2018-08-08 | 2018-12-07 | 王冬国 | Application of the LOC105369370 in rectal adenocarcinoma is diagnosed, treated |
| CN109735622A (en) * | 2019-03-07 | 2019-05-10 | 天津市第三中心医院 | LncRNA relevant to colorectal cancer and its application |
| CN113699234A (en) * | 2021-07-26 | 2021-11-26 | 北京化工大学 | Application of long-chain non-coding RNA Linc01605 in development of gastric cancer diagnostic kit and targeted drugs |
| WO2023143521A1 (en) * | 2022-01-28 | 2023-08-03 | City University Of Hong Kong | Methods and systems for measuring multiplex rna expression |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146688A (en) * | 2012-09-12 | 2013-06-12 | 上海长海医院 | Application of long-chain non-coding RNA as blood molecular marker for disease diagnosis |
| CN103952474A (en) * | 2014-03-27 | 2014-07-30 | 南京市第一医院 | Esophageal carcinoma (EC) diagnosis marker and application method thereof |
| CN104388556A (en) * | 2014-11-11 | 2015-03-04 | 中国人民解放军第二军医大学 | Application of long non-coding RNA in preparation of medicament for diagnosing or treating ovarian cancer |
-
2015
- 2015-05-13 CN CN201510242287.3A patent/CN104877998B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146688A (en) * | 2012-09-12 | 2013-06-12 | 上海长海医院 | Application of long-chain non-coding RNA as blood molecular marker for disease diagnosis |
| CN103952474A (en) * | 2014-03-27 | 2014-07-30 | 南京市第一医院 | Esophageal carcinoma (EC) diagnosis marker and application method thereof |
| CN104388556A (en) * | 2014-11-11 | 2015-03-04 | 中国人民解放军第二军医大学 | Application of long non-coding RNA in preparation of medicament for diagnosing or treating ovarian cancer |
Non-Patent Citations (3)
| Title |
|---|
| FENG YANG1 ET AL: "Long non-coding RNA GHET1 promotes gastric carcinoma cell proliferation by increasing c-Myc mRNA stability", 《FEBS JOURNAL》 * |
| HONG-TU ZHENG ET AL.: "Long non-coding RNA expression profile in human gastric cancer and its clinical significances", 《JOURNAL OF TRANSLATIONAL MEDICINE》 * |
| PAN YANGLIN ET AL: "KCNE2, a down-regulated gene identified by in silico analysis,suppressed proliferation of gastric cancer cells", 《CANCER LETTERS》 * |
Cited By (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106676191B (en) * | 2017-03-03 | 2019-06-21 | 中日友好医院 | A kind of molecular marker for adenocarcinoma of colon |
| CN106676191A (en) * | 2017-03-03 | 2017-05-17 | 中日友好医院 | Molecular marker for colon adenocarcinoma |
| CN107119125A (en) * | 2017-05-11 | 2017-09-01 | 青岛大学 | The application of lncRNA D63785 and its chaff interference and apply its product |
| CN107119125B (en) * | 2017-05-11 | 2020-10-16 | 青岛大学 | Application of IncRNA-D63785 and interferent thereof and product using IncRNA-D63785 and lncRNA-D63785 |
| CN107043823A (en) * | 2017-05-26 | 2017-08-15 | 郴州市第人民医院 | A kind of related tumor markers of colorectal cancer and application |
| CN107043823B (en) * | 2017-05-26 | 2020-05-12 | 郴州市第一人民医院 | Tumor marker related to colorectal cancer and application |
| CN108034655A (en) * | 2017-10-26 | 2018-05-15 | 南京医科大学第二附属医院 | A kind of application of the long non-coding RNA and combinations thereof in diagnosis/treatment colorectal cancer |
| CN108034655B (en) * | 2017-10-26 | 2021-04-27 | 南京医科大学第二附属医院 | Application of long non-coding RNA and composition thereof in diagnosis/treatment of colorectal cancer |
| CN108753960A (en) * | 2018-04-24 | 2018-11-06 | 郑州大学第附属医院 | A kind of gastric cancer prognosis molecule marker non-coding RNA Lnc-DENR-2 and its application |
| CN108949991A (en) * | 2018-08-08 | 2018-12-07 | 王冬国 | Application of the LOC105369370 in rectal adenocarcinoma is diagnosed, treated |
| CN108841964A (en) * | 2018-08-08 | 2018-11-20 | 王冬国 | Application of the LOC105370108 in rectal adenocarcinoma diagnosis and treatment |
| CN108841964B (en) * | 2018-08-08 | 2021-07-02 | 王冬国 | Application of LOC105370108 in diagnosis and treatment of rectal adenocarcinoma |
| CN108949991B (en) * | 2018-08-08 | 2021-07-02 | 王冬国 | Application of LOC105369370 in diagnosis and treatment of rectal adenocarcinoma |
| CN108866198B (en) * | 2018-08-31 | 2020-08-11 | 青岛泱深生物医药有限公司 | Application of marker LOC105376380 in diagnosis and treatment of rectal adenocarcinoma |
| CN108866198A (en) * | 2018-08-31 | 2018-11-23 | 北京泱深生物信息技术有限公司 | Application of the marker LOC105376380 in rectal adenocarcinoma diagnosing and treating |
| CN109735622A (en) * | 2019-03-07 | 2019-05-10 | 天津市第三中心医院 | LncRNA relevant to colorectal cancer and its application |
| CN113699234A (en) * | 2021-07-26 | 2021-11-26 | 北京化工大学 | Application of long-chain non-coding RNA Linc01605 in development of gastric cancer diagnostic kit and targeted drugs |
| CN113699234B (en) * | 2021-07-26 | 2024-03-26 | 北京化工大学 | Application of long-chain non-coding RNA Linc01605 as gastric cancer diagnostic kit and targeted drug development |
| WO2023143521A1 (en) * | 2022-01-28 | 2023-08-03 | City University Of Hong Kong | Methods and systems for measuring multiplex rna expression |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104877998B (en) | 2018-03-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104877998A (en) | Long noncoding RNA (LncRNA), and primer pair and kit for detecting expression level of long noncoding RNA in cells and tissues | |
| Kamel et al. | Investigation of long noncoding RNAs expression profile as potential serum biomarkers in patients with hepatocellular carcinoma | |
| Liu et al. | Clinical significance of the low expression of FER1L4 in gastric cancer patients | |
| AU2013281355B2 (en) | Targeted RNA-seq methods and materials for the diagnosis of prostate cancer | |
| Ding et al. | Promoter methylation and mRNA expression of DKK-3 and WIF-1 in hepatocellular carcinoma | |
| CN105316341B (en) | A kind of LncRNA and its application in marker or prostate cancer prognosis recurrence marker is detected as prostate cancer | |
| Zeng et al. | Polymorphisms in prostate stem cell antigen gene rs2294008 increase gastric cancer risk in Chinese | |
| CN109055563B (en) | Colorectal cancer related circular RNA gene, colorectal cancer molecular marker and application thereof | |
| Wen et al. | Identification of two microRNA signatures in whole blood as novel biomarkers for diagnosis of nasopharyngeal carcinoma | |
| CN104131108A (en) | LncRNA biomarkers for diagnosing human lung adenocarcinoma and human colorectal cancer | |
| US10214778B2 (en) | Methods and nucleotide fragments of predicting occurrence, metastasis of cancers and patients' postoperative survival in vitro | |
| Kulda et al. | Prognostic significance of TMPRSS2-ERG fusion gene in prostate cancer | |
| CN106811525B (en) | Kit and system for predicting early postoperative recurrence of stage III gastric cancer patient | |
| Han et al. | Diagnostic value of four serum exosome microRNAs panel for the detection of colorectal cancer | |
| CN105802964B (en) | The detection and its application of a kind of cRNA-FUT8 for hepatocellular carcinoma examination | |
| CN104498495A (en) | Prostatic cancer marker lncRNA MALAT-1 and application thereof | |
| Wu et al. | Identification of a long non-coding RNA NR_026689 associated with lung carcinogenesis induced by NNK | |
| CN104878008A (en) | Long noncoding RNA, primer pair for detecting expression level of long noncoding RNA in cell line and tissue as well as kit | |
| Kang et al. | Comparison of PNA clamping and direct sequencing for detecting KRAS mutations in matched tumour tissue, cell block, pleural effusion and serum from patients with malignant pleural effusion | |
| CN109022583A (en) | Hsa_circ_0021977 is preparing the application on Diagnosis of Breast cancer product | |
| CN102851354B (en) | Primer pair and kit for detecting methylation state of DNA at cell DACH1 gene promoter region | |
| CN105567797A (en) | PCR primer, method and kit for detecting CpG island methylation of DACT2 gene promoter area | |
| Zhang et al. | A panel of differentially methylated regions enable prognosis prediction for colorectal cancer | |
| Feng et al. | Analysis of CASP12 diagnostic and prognostic values in cervical cancer based on TCGA database | |
| CN104711368A (en) | Noninvasive polygenic hereditary colorectal cancer detection kit based on next-generation sequencing technology |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |