CN108753960A - A kind of gastric cancer prognosis molecule marker non-coding RNA Lnc-DENR-2 and its application - Google Patents
A kind of gastric cancer prognosis molecule marker non-coding RNA Lnc-DENR-2 and its application Download PDFInfo
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Abstract
The present invention relates to gastric cancer prognosis molecule marker non-coding RNA Lnc-DENR-2 and its applications, it can effectively solve the problems, such as to prepare the kit for predicting patients with gastric cancer prognosis, a kind of gastric cancer prognosis molecule marker non-coding RNA Lnc-DENR-2, its transcript length is more than 200 nucleotide, and sequence is SEQ NO:1, the application in preparing gastric cancer prognosis real-time fluorescence quantitative PCR detection kit, the kit includes the specific primer for the kit expression for detecting patients with gastric cancer prognosis being placed in box body, and primer sequence is SEQ No：2 and 3, the primer sequence of reference gene TUBA1A expression, primer sequence are SEQ No：4,5 and RNA is extracted from gastric tissue, and carry out the reagent of reverse transcription and quantitative fluorescent PCR, molecular marker new as gastric cancer prognosis non-coding RNA Lnc-DENR-2 of the present invention, gastric cancer prognosis can be predicted in time, and patients with gastric cancer is treated in time, extend the survival of patients time, improve survival rate, is the big innovation in gastric cancer prognosis.
Description
Technical field
The present invention relates to oncomolecularbiology, especially a kind of gastric cancer prognosis molecule marker non-coding RNA Lnc-
DENR-2 and its application.
Background technology
Primary Hepatic cancer morbidity occupies the third position of world's tumor incidence, the main reason for being whole world cancer mortality.
Its early diagnostic rate is low, and whens many case-findings has lost operation cutting opportunity, and liver cancer has the high rate of transform and recurrence rate,
Poor prognosis, the death rate are high.Although have developed rapidly for current medical technology, is on the increase, still for the treatment means of liver cancer
Fail the poor prognosis of change liver cancer.How prior involvement intervention during liver cancer genesis and development, prevent trouble before it happens, to pre-
Anti- or inhibition liver cancer is of great significance.Currently, clinically there are no ideal molecular indexes come assess prognosis and
The risk shifted.Therefore, it is current urgent problem to find new prognosis biomarker.
Hrr gene chip and mankind's genome sequencing are analysis shows that it includes about 20000 to go out human genome
A encoding egg white gene.However most of human genome includes a large amount of non-coding RNA, these non-coding sequences are in people
The accounting of genoid group is more than 98%.Non-coding RNA can simply be divided into two classifications, i.e. long-chain non-coding according to their size
RNA and tiny RNA.Long-chain non-coding RNA is as a kind of novel non-coding RNA, and length is more than 200 nucleotide, not due to it
Has coding protein function, originally they are considered not having biological function, but " noise or rubbish in subgenomic transcription
Rubbish ".Since long-chain non-coding RNA is compared with microRNA, have structured complexity and the diversity of expression regulation, and grind
Study carefully and finds that long-chain non-coding RNAs play the role of that some are very important in epigenetic modification, pass through control and encode base
Because of the transcription of upstream promoter, inhibit the chromatinic recombination of the activity of RNA polymerase, Jie's structure, the shearing of interference mRNA and albumen
The modes such as the positioning of matter Binding change protein in the cell influence the expression of gene, diversified in organism to participate in
Pathophysiological process.Lnc-DENR-2 is a kind of newfound non-coding RNA, and transcript length is more than 200 nucleotide.
Currently, the important function that non-coding RNA Lnc-DENR-2 is played during normal cell development or tumor development is also
In further research.Whether non-coding RNA Lnc-DENR-2 can be as a kind of new tumor markers reagent preparation and examination
Agent box is applied to prediction patients with gastric cancer prognosis so far there are no open report.
Invention content
For the above situation, to overcome the defect of the prior art, the purpose of the present invention to be just to provide a kind of gastric cancer prognosis point
Sub- marker non-coding RNA Lnc-DENR-2 and its application can effectively solve the kit for preparing prediction patients with gastric cancer prognosis
Problem.
The technical solution that the present invention solves is a kind of gastric cancer prognosis molecule marker non-coding RNA Lnc-DENR-2,
Transcript length is more than 200 nucleotide, and sequence is SEQ NO:1；
The gastric cancer prognosis molecule marker non-coding RNA Lnc-DENR-2 is preparing the detection of gastric cancer prognosis real-time fluorescence quantitative PCR
Application in kit；
The gastric cancer prognosis real-time fluorescence quantitative PCR detection kit include be placed in box body for detect patients with gastric cancer pre-
The specific primer of kit expression afterwards, primer sequence are SEQ No：2 and 3, the primer sequence of reference gene TUBA1A expression
Row, primer sequence are SEQ No：4,5 and RNA is extracted from gastric tissue, and carry out the reagent of reverse transcription and quantitative fluorescent PCR.
Lnc-DENR-2 of the present invention is a kind of newfound non-coding RNA, can be effectively used for preparing patients with gastric cancer prognosis
Kit, molecular marker new as gastric cancer prognosis non-coding RNA Lnc-DENR-2 can predict gastric cancer prognosis in time, and and
When patients with gastric cancer is treated, extend the survival of patients time, improve survival rate, be the big innovation in gastric cancer prognosis, it is economical
It is huge with social benefit.
Description of the drawings
Fig. 1 is that real-time fluorescence quantitative PCR of the present invention analyzes differential expressions of the Lnc-DENR-2 in normal structure and gastric cancer
Figure.
Fig. 2 is the Lnc-DENR-2 tables that the present invention analyzes the influence prognosis survivorship curve of patients with gastric cancer in stomach organization source
Up to high-low graph.
Specific implementation mode
It elaborates to the specific implementation mode of the present invention below in conjunction with concrete condition.
In specific implementation, a kind of gastric cancer prognosis molecule marker non-coding RNA Lnc-DENR-2 is sent out the present invention to be new
A kind of existing non-coding RNA Lnc-DENR-2 molecular markers, are long-chain non-coding RNA, and transcript length is more than 200
Nucleotide, the nucleotides sequence are classified as SEQ No：1：
SEQ NO:1
TGAAGTGGTACCTTCTTTAGAAAGAGAAGAACGTTTAACAAGGAAGAAAAGGGCCAACCTGCAGCGAGATTTG
GAGAAGGGAAACAGCATTTGCAGGACATCCACTCAACTCTAGGCTTCGTGCCAGGCGGCTTAAGTATATGATCACAG
CCTCGGCTTGGGAGGGGCCTGAGGGCCTAATGAACAGCCTGGTACAGCCTGGTACAGCCTAGGACATCCTGGGAAGT
TCTGGAAATACTGACTCCAAGCAGGTCAAGTGTGGAGTAGTTTTCTACCAGGACAGAGTTAACAACCCATAAAGGTC
TCTGCTTAAGTAAGAAGTCCATTTCTGTGAACCATAACCCAGATATAGCAACGCCAGACTGTCTCCTATCAAAATTT
ATTGAAATGGTAGATTTTTGTGTGGAGTAGTTTTCTACCAGGACAAAGTTAACAACCCATAAAGGTCTCTGCTTAAG
TAAGAAGTCCATTTCTGTGAACCATAACCCAGATATAGCAACGCCAGACTGTCTCCTATCAAAATTTATTGAAATGG
TAGATTTTTGGTAAGTACAGAAACACAAGCGAAGGAACCAGGAGGAACGTGCACCCCTCTACCCGTGGGGCAGAGGC
ACGTTTCCCTTTTAATGGTAAAAGCAAACAGGTGGGAAACAGCTTTCTATCCTGAAGCCGTTTTAACTGTTCAACCC
TTAGATTACTATAAACAAGAGAGGAATGGATTGCATTAGTCGCTGGTTGAAAGCAGCAATGCCATTTCCTTTCCCAT
AATAACACTGTTTAGCTCAGTCCCTTCTGGGGCTAATCCCCTATTACACCCACCGGAAACACAGATACTAACCAAAG
CATGTGAGTCTCTTCCCTGAGTCCATTTCTGCTAAAGATTGTCTGAAAGATGAATTGGTCCACGGAAGCCAGGTGAC
CTACTGGCTTGTTTAATGATGCCTGGGCTTCCTCCAGGAACAACACAATTCCCTCCAATCTCAGCTCTCCTTATCCC
TGCTAGAGCTCACAAGCAGGCTAGATATCACCCCAGGAGCTGAGCCCCCTCCAGTCTGAGGCAGATGTGGGAAGAAG
GCCAACAAGTCACAAGTAGATCTCTGGCTCCAACTCTGCTCAGCAGAAACGCCTACAATTTGCCCATCTTCAGTCCT
GCGAGCGTCAGAGATGAAGCAAGTTTCAGATGCCTAGAAGCTTTACTCTCTGTTCCTCCAGGATTCCTGCGGTCACA
CCTTGCAACCAGTCTCCCACTCATCTGCCACAGTTTCCCTAAATCAGATTCTCTGAATCTGGAAGTTCCAGGAAATC
TTAGGCCGAGTCCAGTGACATTACTCGATGCAACAGCCCAACTGTTTCTCCAGAGATGCTGGTTCTTGGTGACAATG
TCCCTTCTTGGAATGGTTATTTGAGGTTGGGCCCAGATAAGAGGGGCTCCATGGCTCACCGGAGTTGGCGATTAACG
CCTCTGGAGCGCCTCTGGTTTTGTTGGGGTCCCCTGTGAGCTCGACGCTCGTGCTGCGGTTATTATCTGGCTCACCT
GTTATCTTCCTCTGGAGGCAGCGGTTGATCAAAGTGGAGAAGAAGTTGGGAAAGGATGGGCTGGAGAAGTAGTACAC
CACGGGGTCCAGCATGCTGTTCATGTAGGTGAAGCTGAGAGTGATAAAGAACGCCAGGTCCACCGAGCGGTACACTT
CACAATTCTGCGTGCCCGAAGTGTGCAGGAGCCAGAAGATG 1731；
The gastric cancer prognosis molecule marker non-coding RNA Lnc-DENR-2 is preparing the detection of gastric cancer prognosis real-time fluorescence quantitative PCR
Application in kit；
The gastric cancer prognosis real-time fluorescence quantitative PCR detection kit include be placed in box body for detect patients with gastric cancer pre-
The specific primer of kit expression afterwards, primer sequence are SEQ No：2 and 3, the primer sequence of reference gene TUBA1A expression
Row, primer sequence are SEQ No：4,5 and RNA is extracted from gastric tissue, and carry out the reagent of reverse transcription and quantitative fluorescent PCR,
In：
SEQ NO:2
GGTTGAAAGCAGCAATGCCA
SEQ NO:3
AACTGTGGCAGATGAGTGGG
SEQ NO:4
gccaaacgtgcctttgttca
SEQ NO:5
gttgcttcttacagcgcgac
The kit of Lnc-DENR-2 expression quantity is that real-time fluorescence determines PCR detection kit in the detection stomach organization.
The real-time fluorescence quantitative PCR detection kit includes the specific primer for carrying out real-time fluorescence quantitative PCR, packet
The specific primer for detecting Lnc-DENR-2 expression is included, primer sequence is SEQ NO:2,3 and reference gene TUBA1A
The primer sequence of expression, primer sequence are SEQ NO: 4,5.
The real-time fluorescence quantitative PCR detection kit also contains in addition to the primer of Lnc-DENR-2 from stomach organization
Middle extraction RNA and all reagents for carrying out reverse transcription and quantitative fluorescent PCR.Including：
(1) the extracted total RNA agents useful for same from the tumour of liver cancer or normal control tissue, including Trizol liquid, chloroform, isopropyl
Alcohol inhibits RNA degradations solvent, 75% ethyl alcohol, DEPC water；
(2) it is cDNA agents useful for same by Lnc-DENR-2 reverse transcriptions by template of total serum IgE, including reverse transcription reaction buffer solution, contains
Have and contains Mg2+Triphosphoric acid base deoxynucleotide dNTP, reverse transcriptase M-MLV and random primer and Oligo dT primers it is mixed
Close object；
(3) by cDNA real-time quantitative PCR agents useful for same, including Lnc-DENR-2 real-time fluorescence quantitative PCRs specific primer,
TUBA1A internal references Specific PCR primers, real time fluorescent quantitative SYBR dyestuffs, the water without RNA enzyme.
The present invention has found the Lnc-DENR-2 and patient in liver cancer tissue source by quantitative fluorescent PCR and survivorship curve analysis
Survival rate it is related, content is higher, and survival rate is higher.This method provides for the prognosis life cycle analysis of prediction liver cancer patient to be had by force
The technical support of power helps to improve the postoperative life quality of liver cancer patient, works out aftertreatment scheme, improves survival rate, tool
There is far-reaching clinical meaning.And through site test, very satisfied advantageous effects are achieved, relevant information is as follows：
The reagent that embodiment 1 prepares detection Lnc-DENR-2 expression quantity is used to prepare the kit of liver cancer patient prognosis (for 30
Secondary response)
1. Trizo l20ml
2. inhibiting RNA degradation solvents 40ml
3. chloroform 80ml
4. isopropanol 80ml
5. DEPC water 10ml
6. the 100 μ l of mixture of 10 × random primer and Oligo dT primers
7. 5 × reverse transcription reaction buffer solution, 150 μ l
8. 10mM contains Mg2+100 μ l of triphosphoric acid base deoxynucleotide dNTP
9. 50 μ l of 200U/ μ l M-MLV reverse transcriptases
10. SYBR Green qPCR Mix 500μl
11. 3 μM of target gene Lnc-DENR-2 specific primers（Its sequence such as SEQ NO:2, shown in 3） 50μl
12. 3 μM of reference gene TUBA1A specific primers（Its sequence such as SEQ NO:4, shown in 5） 50μl
The detection of 2 tissue samples Lnc-DENR-2 of embodiment
1, liver cancer to be measured or normal control tissue are collected, after physiological saline cleans up, is put into and fills inhibition RNA degradation solvents
In cryopreservation tube, put spare to -80 DEG C of refrigerators.
2, the extracting of RNA in organizing：
（1）Liquid nitrogen is first added in mortar, then tissue is cut into small pieces and is clayed into power in liquid nitrogen, is taken with the spoon of Liquid nitrogen precooler
100mg organizes powder to be added in the EP pipes for the Trizol liquid for having filled 1ml.Organize total powder volume no more than Trizol used
The 10% of volume is sufficiently mixed uniformly.
（2）5 minutes are placed at room temperature for, the chloroform of 200 μ L is then added, EP is covered tightly and manages and acutely sway 0.5 minute.12000
Rev/min centrifugation 10 minutes.
（3）Take upper strata aqueous phase in a new EP pipes（The intermediate beds of precipitation and subnatant are not mixed into）, 500 μ L isopropyls are added
Alcohol mildly overturns mixing.10 minutes are placed at room temperature for, 12000 revs/min centrifuge 10 minutes.
（4）Liquid is carefully discarded supernatant, is added 75% ethyl alcohol of 1ml, vortex mixing, 12000 revs/min of centrifugations 5 at 4 DEG C
Minute.Repetitive operation is primary.
（5）Discard supernatant liquid（Residual liquid is removed as possible）, room temperature or vacuum drying 5~10 minutes（It is careful not to do
It is dry excessive, it otherwise can reduce the solubility of RNA）.RNA is dissolved with 50 μ L DEPC processed water.
（6）RNA concentration mensurations：It is measured with nucleic acid concentration analyzer, inhales 1 μ L RNA samples and add to sample well, according to
Reading directly determines the concentration of RNA.The OD260/OD280 ratios of the measurement of nucleic acid concentration analyzer, ratio is between 1.8-2.0
Think that RNA purity is fine.Finally, it is spare to be stored in -80 DEG C of refrigerators by RNA.
3, the reverse transcription of Lnc-DENR-2 RNA：Use the Reverse Transcriptase kit of the green skies Bioisystech Co., Ltd in Shanghai
（D7170S）.It is as follows.
（1）Remove genomic DNA：Template Total RNA, 5 × gDNA Eraser Buffer are thawed on ice, 5 ×
RT Buffer, 10 × RT Primer Mix, DEPC-treated Water are in (15-25 DEG C) defrosting of room temperature, after defrosting rapidly
It is placed on ice.Using it is preceding by each solution mixing and it is of short duration centrifugation so that all liq is settled down to tube bottom.The denaturation of RNA,
RNA sample thermal denaturation under the conditions of 65 DEG C is immediately placed in ice water cooling after 5 minutes.According to the form below ingredient is in preparation reaction on ice
Then mixed liquor is dispensed into each reaction tube, is eventually adding RNA sample again.
| Reagent | Usage amount |
| 5×gDNA Eraser Buffer | 2μl |
| Total RNA | up to 0.5μg |
| DEPC-treated Water | To 10 μ l |
| Total volume | 10μl |
（2）In PCR instrument or in water-bath, 37 DEG C are incubated 2 minutes.Be immediately placed in place on ice it is spare.
（3）Reverse transcription system is prepared：Reaction solution preparation is carried out on ice, and reverse transcription reaction is carried out immediately after soft mixing.
Mixed liquor is prepared according to the reverse transcription reaction system of following table.
| Reagent | Usage amount |
| 5×RT Buffer（Containing Mg2+And dNTP） | 4μl |
| 10×RT Primer Mix（Oligo dT Primer and Random Hexamers mixtures） | 2μl |
| BeyoRT II M-MLV reverse transcriptases (RNase H-)（Inhibitor containing RNase） | 2μl |
| DEPC-treated water | 2μl |
| Total RNA after above-mentioned removal gDNA | 10μl |
| Total volume | 20μl |
（4）42 DEG C are incubated 60 minutes to carry out reverse transcription reaction, after subsequent 80 DEG C are incubated 10 minutes inactivation reverse transcriptases
It is put on ice.For the template of secondary structure complexity or high GC content, reverse transcription temperature can be improved to 50 DEG C, reversed with enhancing
Record efficiency.
（5）Obtained cDNA can immediately or -80 DEG C freeze after be used for follow-up real-time fluorescence quantitative PCR, cDNA preferably avoided
More multigelations.
4, real-time quantitative PCR is carried out using the specific primer of Lnc-DENR-2：Specific primer is in raw work bioengineering
（Shanghai）Limited liability company synthesizes, and includes the specific primer for detecting Lnc-DENR-2 expression, primer sequence SEQ
NO:2, the primer sequence that 3 and reference gene TUBA1A is expressed, primer sequence are SEQ NO: 4,5.Real-time quantitative PCR its
Its reagent utilizes the BeyoFast SYBR Green qPCR Mix (2 ×) of the green skies Bioisystech Co., Ltd in Shanghai, tool
Steps are as follows for body.
（1）Melt and mixing PCR reacts required various solution, BeyoFast SYBR Green qPCR Mix are complete
Melt and mixing is placed in ice chest.
（2）PCR reaction systems (by taking 96 orifice plates as an example) are set on ice bath：
| Reagent | Usage amount |
| BeyoFast™ SYBR Green qPCR Mix (2X, Low ROX) | 10μl |
| Specific primer (3 μM of concentration) | 2μl |
| Template DNA | 2μl |
| Water without RNA enzyme | 6μl |
| Total volume | 20μl |
(3) the usually amount of DNA profiling with 1-10ng cDNA is with reference to dosage, and final concentration of 0.2-0.5 μM of primer when can be obtained
Good detection result is obtained, the final concentration of primer also can be according to circumstances adjusted.If it is necessary, gradient dilution can be carried out to template,
With the template usage amount that determination is best.When reverse transcription PCR cDNA obtained by the reaction is directly as template, additive amount does not exceed
PCR reacts the 10% of total volume.The recommendation response system of 96 orifice plates is 20 μ l, can also in proportion be expanded according to actual experiment demand
Big or diminution reaction system.
(4) mixing or slight Vortex mixings are gently blown and beaten with pipettor, room temperature centrifuges the several seconds, liquid is made to accumulate in pipe
Bottom.
(5) the PCR reaction tubes set or PCR reaction plates are placed on fluorescence quantitative PCR instrument, start PCR reactions.
(6) PCR response procedures：The pre-degeneration that template is carried out before real-time fluorescence quantitative PCR reacts, is typically set at 95
DEG C 2 minutes.Using following PCR programs, this program is by taking ABI 7900HT fluorescence quantitative PCR instruments as an example：
A. pre-degeneration：95℃ 2min
B. it is denaturalized：95℃ 15sec
C. annealing/extension：60℃ 15-30sec
D. step b and step c is repeated, in total 40 cycles
E. melting curve analysis (optional)：95℃ 15sec, 60℃ 15sec, 95℃ 15sec
F. the software analysis result for using fluorescence quantitative PCR instrument to provide
Three-step approach only need to after annealing/extension plus 72 DEG C of 30sec of a step, then repeat step b, c and it is increased the step for it is total
40 cycles.
5, the data analysis of Lnc-DENR-2 expression quantity：This experimental data is included in 60 liver cancer patients and its Normal group
It knits.The interpretation of result of real-time quantitative PCR uses relative quantitation method, that is, 2^-△△CtMethod.It is specific as follows：First, it will once test
All gene C t values are put in order, are subtracted in itself with the Ct values of the target gene Lnc-DENR-2 in each group of tumor sample later
Join the Ct values of gene TUBA1A, obtained number is exactly △ Ct；Changing formula into is exactly：△Ct=Ct（Target gene Lnc-DENR-2）-
Ct（Reference gene TUBA1A）；Then, the △ Ct of each group of each target gene of tumor sample Lnc-DENR-2 are calculated.
The △ Ct of normal control tissue group sample are subtracted with the △ Ct of tumor tissues sample in this experiment, and all results are taken simultaneously
Opposite number, the result which obtains are exactly-△ △ Ct.Finally, p- △ △ Ct carry out 2 power operation, i.e. 2^-△△CtIt must
The multiple for going out expression quantity changes.In triplicate, it is examined using nonparametric t- for statistical analysis.It was found that the tumour of liver cancer
The expression quantity of Lnc-DENR-2 is higher than normal control in tissue（See Fig. 1）, difference have significant difference (p< 0.05).
6, by 60 liver cancer patient follow-up statistics being included in above-mentioned experiment, including patient's First episode when
Between, treatment, recurrence status and death time etc., follow up time is at least 12 months.In selected liver cancer patient, choosing
It is reference standard to take the expression value that fluorescence real-time quantitative PCR is analyzed, and the corresponding normal structure of acquired results compares.Tumour
Lnc-DENR-2 expression is defined as Lnc-DENR-2 high expression groups higher than the patient of normal control tissue in tissue, remaining is low table
Up to group.By Kaplan-Meier survival analysis, Lnc-DENR-2 high expresses the life cycle ratio Lnc-DENR-2 low expressions of patient
The patient of group is substantially reduced, and prognosis is worse（See Fig. 2）, difference have it is statistically significant (p< 0.05).Therefore, Lnc-DENR-
2 can be used as the specificity molecular marker of liver cancer patient prognosis.
By above it should be apparent that the invention discloses a kind of new gastric cancer prognosis molecule marker non-coding RNAs
Lnc-DENR-2 is used to prepare the kit of patients with gastric cancer, by the examination for detecting the tissue-derived Lnc-DENR-2 of gastric cancer tumor
Agent box.Lnc-DENR-2 up-regulated expressions in gastric cancer, the patients with gastric cancer of height expression Lnc-DENR-2, totality are confirmed by research
Survival rate is worse.It therefore, can be to patients with gastric cancer by detecting the expression of Lnc-DENR-2 in patients with gastric cancer tumor tissues
Prognosis inspection diagnosis is made, accuracy rate is up to 97% or more, and is treated in time, and survival and life quality, tool are improved
There are far-reaching clinical application significance and application value, economic and social benefit huge.
Sequence table
<110>The first affiliated hospital of Zhengzhou University
<120>A kind of gastric cancer prognosis molecule marker non-coding RNA Lnc-DENR-2 and its application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1731
<212> DNA
<213> Homo sapiens
<400> 1
tgaagtggta ccttctttag aaagagaaga acgtttaaca aggaagaaaa gggccaacct 60
gcagcgagat ttggagaagg gaaacagcat ttgcaggaca tccactcaac tctaggcttc 120
gtgccaggcg gcttaagtat atgatcacag cctcggcttg ggaggggcct gagggcctaa 180
tgaacagcct ggtacagcct ggtacagcct aggacatcct gggaagttct ggaaatactg 240
actccaagca ggtcaagtgt ggagtagttt tctaccagga cagagttaac aacccataaa 300
ggtctctgct taagtaagaa gtccatttct gtgaaccata acccagatat agcaacgcca 360
gactgtctcc tatcaaaatt tattgaaatg gtagattttt gtgtggagta gttttctacc 420
aggacaaagt taacaaccca taaaggtctc tgcttaagta agaagtccat ttctgtgaac 480
cataacccag atatagcaac gccagactgt ctcctatcaa aatttattga aatggtagat 540
ttttggtaag tacagaaaca caagcgaagg aaccaggagg aacgtgcacc cctctacccg 600
tggggcagag gcacgtttcc cttttaatgg taaaagcaaa caggtgggaa acagctttct 660
atcctgaagc cgttttaact gttcaaccct tagattacta taaacaagag aggaatggat 720
tgcattagtc gctggttgaa agcagcaatg ccatttcctt tcccataata acactgttta 780
gctcagtccc ttctggggct aatcccctat tacacccacc ggaaacacag atactaacca 840
aagcatgtga gtctcttccc tgagtccatt tctgctaaag attgtctgaa agatgaattg 900
gtccacggaa gccaggtgac ctactggctt gtttaatgat gcctgggctt cctccaggaa 960
caacacaatt ccctccaatc tcagctctcc ttatccctgc tagagctcac aagcaggcta 1020
gatatcaccc caggagctga gccccctcca gtctgaggca gatgtgggaa gaaggccaac 1080
aagtcacaag tagatctctg gctccaactc tgctcagcag aaacgcctac aatttgccca 1140
tcttcagtcc tgcgagcgtc agagatgaag caagtttcag atgcctagaa gctttactct 1200
ctgttcctcc aggattcctg cggtcacacc ttgcaaccag tctcccactc atctgccaca 1260
gtttccctaa atcagattct ctgaatctgg aagttccagg aaatcttagg ccgagtccag 1320
tgacattact cgatgcaaca gcccaactgt ttctccagag atgctggttc ttggtgacaa 1380
tgtcccttct tggaatggtt atttgaggtt gggcccagat aagaggggct ccatggctca 1440
ccggagttgg cgattaacgc ctctggagcg cctctggttt tgttggggtc ccctgtgagc 1500
tcgacgctcg tgctgcggtt attatctggc tcacctgtta tcttcctctg gaggcagcgg 1560
ttgatcaaag tggagaagaa gttgggaaag gatgggctgg agaagtagta caccacgggg 1620
tccagcatgc tgttcatgta ggtgaagctg agagtgataa agaacgccag gtccaccgag 1680
cggtacactt cacaattctg cgtgcccgaa gtgtgcagga gccagaagat g 1731
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 2
ggttgaaagc agcaatgcca 20
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 3
aactgtggca gatgagtggg 20
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 4
gccaaacgtg cctttgttca 20
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 5
gttgcttctt acagcgcgac 20
Claims (3)
1. a kind of gastric cancer prognosis molecule marker non-coding RNA Lnc-DENR-2 is long-chain non-coding RNA, transcript length
More than 200 nucleotide, the nucleotides sequence are classified as SEQ No：1.
2. gastric cancer prognosis molecule marker non-coding RNA Lnc-DENR-2 described in claim 1 is real-time in preparation gastric cancer prognosis
Application in fluorescent quantificationally PCR detecting kit.
3. the gastric cancer prognosis molecule marker non-coding RNA Lnc-DENR-2 described in claim 2 is real-time in preparation gastric cancer prognosis
Application in fluorescent quantificationally PCR detecting kit, which is characterized in that the gastric cancer prognosis real-time fluorescence quantitative PCR detection examination
Agent box includes the specific primer for the kit expression for detecting patients with gastric cancer prognosis being placed in box body, and primer sequence is
SEQ No：2 and 3, the primer sequence of reference gene TUBA1A expression, primer sequence are SEQ No：4, it 5 and is extracted from gastric tissue
RNA, and carry out the reagent of reverse transcription and quantitative fluorescent PCR.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104877998A (en) * | 2015-05-13 | 2015-09-02 | 中国人民解放军总医院 | Long noncoding RNA (LncRNA), and primer pair and kit for detecting expression level of long noncoding RNA in cells and tissues |
| CN105018639A (en) * | 2015-08-25 | 2015-11-04 | 王振宁 | Detection and application of novel molecular marker IncRNA (long non-coding RNA) AB007962 for gastric cancer prognosis |
| CN105200156A (en) * | 2015-10-30 | 2015-12-30 | 中南大学 | Application of long-chain non-coding RNA (ribonucleic acid) gene LOC553103 |
-
2018
- 2018-04-24 CN CN201810373549.3A patent/CN108753960A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104877998A (en) * | 2015-05-13 | 2015-09-02 | 中国人民解放军总医院 | Long noncoding RNA (LncRNA), and primer pair and kit for detecting expression level of long noncoding RNA in cells and tissues |
| CN105018639A (en) * | 2015-08-25 | 2015-11-04 | 王振宁 | Detection and application of novel molecular marker IncRNA (long non-coding RNA) AB007962 for gastric cancer prognosis |
| CN105200156A (en) * | 2015-10-30 | 2015-12-30 | 中南大学 | Application of long-chain non-coding RNA (ribonucleic acid) gene LOC553103 |
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Application publication date: 20181106 |