CN108424965A - A kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc-CRYBA4-23 and its application - Google Patents
A kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc-CRYBA4-23 and its application Download PDFInfo
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Abstract
The present invention relates to cancer of pancreas prognosis molecule marker non-coding RNA Lnc CRYBA4 23 and its applications, it can effectively solve the problems, such as to prepare the kit for predicting Pancreas cancer patients prognosis, a kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc CRYBA4 23, its transcript length is more than 200 nucleotide, and sequence is SEQ NO:1, the application in preparing cancer of pancreas prognosis real-time fluorescence quantitative PCR detection kit, the kit includes the specific primer for the kit expression for detecting Pancreas cancer patients 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 and 5 and RNA is extracted from Pancreatic Adenocarcinoma, and carry out the reagent of reverse transcription and quantitative fluorescent PCR.Molecular marker new as cancer of pancreas prognosis non-coding RNA Lnc CRYBA4 23 of the present invention, can predict cancer of pancreas prognosis, and treated in time to Pancreas cancer patients in time, extend the survival of patients time, improve survival rate, be the big innovation in cancer of pancreas prognosis.
Description
Technical field
The present invention relates to oncomolecularbiology, especially a kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc-
CRYBA4-23 and its application.
Background technology
Cancer of pancreas is a kind of alimentary system malignant tumour that prognosis is very dangerous.In the past 30 years, incidence is in and rises year by year
Trend.Due to cancer of pancreas insidious onset, no specific symptom and sign, the apparent disease such as yellow subcutaneous ulcer, abdominal pain and lumbago to appear
When shape and sign, mostly to late period, because of tumor by local infiltration, blood vessel is invaded or DISTANT METASTASES IN, loses surgical indication, only closely
20% patient can be with underwent operative tumor resection, and poor prognosis, postoperative 5 years survival rates are less than 10%.Currently, clinically not yet
Ideal molecular indexes are come the risk assessing prognosis and shift.Therefore, it is current urgency to find new prognosis biomarker
It need to solve the problems, such as.
Sequence in human genome only less than 2% is protein coding, although other sequences non-coding protein,
RNA can be transcribed into more than 90%.These cannot be that the RNA molecule of protein coding is referred to as non-coding RNA.Non-coding RNA
Various biological process is taken part in, and is played a significant role in tumor development.Non-coding RNA is roughly divided into based on scale
Two major classes：Short chain non-coding RNA and long-chain non-coding RNA.As the new role in non-coding RNA, long-chain non-coding RNA is each
Important regulating and controlling effect is played in kind disease especially tumour, becomes the hot spot of tumor research.Long-chain non-coding RNA is in a variety of levels
The expression of the controlling genes such as level-off i.e. after epigenetic level, transcriptional level and transcription.A large amount of evidences show that a variety of long-chains are non-
There are unconventionality expressions in multiclass tumour for coding RNA, and rush cancer or cancer suppressing action are played in the generation, evolution of tumour.
In recent years, more and more research shows that the generation of the unconventionality expression of long-chain non-coding RNA and tumour, development and invasion transfer have
Substantial connection.Therefore, explore tumour correlation long-chain non-coding RNA and correlation between protein coding gene at
For the important topic of knubble biological research field.Lnc-CRYBA4-23 is a kind of newfound non-coding RNA, and transcript is long
Degree is more than 200 nucleotide.Currently, non-coding RNA Lnc-CRYBA4-23 is in normal cell development or tumor development
The important function played in the process is also in further research.Whether Lnc-CRYBA4-23 can be as a kind of new tumour mark
Will object reagent preparation and kit are applied to prediction Pancreas cancer patients 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 cancer of pancreas prognosis
Molecular marker non-coding RNA Lnc-CRYBA4-23 and its application can effectively solve to prepare prediction Pancreas cancer patients prognosis
The problem of kit.
The technical solution that the present invention solves is a kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc-CRYBA4-
23, transcript length is more than 200 nucleotide, and sequence is SEQ NO:1；
The cancer of pancreas prognosis molecule marker non-coding RNA Lnc-CRYBA4-23 is preparing cancer of pancreas prognosis real time fluorescent quantitative
Application in PCR detection kit；
The cancer of pancreas prognosis real-time fluorescence quantitative PCR detection kit includes being placed in suffering from for detecting cancer of pancreas in box body
The specific primer of the kit expression of person's prognosis, primer sequence are SEQ No：2 and 3, the primer of reference gene TUBA1A expression
Sequence, primer sequence are SEQ No：4,5 and RNA is extracted from Pancreatic Adenocarcinoma, and carry out reverse transcription and quantitative fluorescent PCR
Reagent.
Lnc-CRYBA4-23 of the present invention is a kind of newfound non-coding RNA, and it is pre- to can be effectively used for preparation Pancreas cancer patients
Kit afterwards, molecular marker new as cancer of pancreas prognosis non-coding RNA Lnc-CRYBA4-23, can predict pancreas in time
Cancer prognosis, and Pancreas cancer patients are treated in time, extend the survival of patients time, improves survival rate, be in cancer of pancreas prognosis
A big innovation, economic and social benefit is huge.
Description of the drawings
Fig. 1 is that real-time fluorescence quantitative PCR of the present invention analyzes expression of the Lnc-CRYBA4-23 in normal structure and cancer of pancreas
Disparity map.
Fig. 2 is the Lnc- that the present invention analyzes the influence prognosis survivorship curve of Pancreas cancer patients in Pancreatic Adenocarcinoma source
CRYBA4-23 expresses 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 cancer of pancreas prognosis molecule marker non-coding RNA Lnc-CRYBA4-23 is the present invention
A kind of newfound non-coding RNA Lnc-CRYBA4-23 molecular markers, are long-chain non-coding RNA, and transcript length is big
In 200 nucleotide, which is classified as SEQ No：1：
SEQ NO:1
CAGGCTGGAGTAGACAGGAACCACGAGAGGCAGGGAGGACTGTACAGGGGTTGCTGACCAGGTGGAAGATGAT
GGGAGCCTCAGAAGGTGTCGTGGGACAGGGTCAGGGAGAGGGCTCTATGCGGCCAGGATCTGGATGCAGATTGTTGA
GACTGAAATTTGGCTCAGATCCCTTCTAGTTGTGTCTGTTTATCTTCAGTTTCCTTGTCTATAAAATGGGGAGAGAT
TGTGTGTCCCCGGCATCACTGGACTGTTAATAGCTATTGCATTTATAGCATTTATCCCAGAACCTGAAACACAGTAA
ATGCTCAGTAAGTGTTATTATTTGCCCCAAACTGTCTTAGTGCTTTCCCCAAGAGCTTTGTGTTGTTTGTACTATGA
GGAATAATGAGGGTGGTGTGGGAGTCGGCCTCTGTGTCTGCAGAGAGGACCACAGGCCACAGGAATAGAGACAGGTA
GATAGGATCAAGGTGACAGGAACAGATGTTCAGAGCAAGAGATGAAGACGGCATCATCAACCCAGACAAGCCAGTTG
GAGGCATCTGTCCACCCATGTGGTTCCAGACACGTTCATGTGGCCACCATGACCCCGTCGGCATCACAGGGGTAACC
AAGAGAGCAGCAAGTTCCTGGGACAGAGGAGACAGAAGGGAATTTCCAGGTTCTTCAGGACGTTCACAACCACACTG
AGAAGCATCTTTGCAGATAAGTATTTAAACTTACCAGCCCATAGCCTACCAGCCTGCCCTCTTCTGGTCTGTGCAGG
AAAGTGTAGTATCCTAGAATGCCAAAGTGGGAGGGGAAATGGGTGATGTAGCTCATTCTCTTTTCTACCTCTATGGA
AGAAAGAAAGAGCCTGTCCCCATTTTGTGGGGTCCCAGAAAGGGTGATTTTCACACTTCACATTTGGCGTTAGGGCT
AGTATTTCACAAACATTACCGTCTGGAACTTTTGAAGGCTGA 962
The cancer of pancreas prognosis molecule marker non-coding RNA Lnc-CRYBA4-23 is preparing cancer of pancreas prognosis real time fluorescent quantitative
Application in PCR detection kit；
The cancer of pancreas prognosis real-time fluorescence quantitative PCR detection kit includes being placed in suffering from for detecting cancer of pancreas in box body
The specific primer of the kit expression of person's prognosis, primer sequence are SEQ No：2 and 3, the primer of reference gene TUBA1A expression
Sequence, primer sequence are SEQ No：4,5 and RNA is extracted from Pancreatic Adenocarcinoma, and carry out reverse transcription and quantitative fluorescent PCR
Reagent, wherein：
SEQ NO:2
GAGAGGCAGGGAGGACTGTA
SEQ NO:3
TTGGTTACCCCTGTGATGCC
SEQ NO:4
actggcttcaaggttggcat
SEQ NO:5
agtgggaggctggtagttga
The kit of Lnc-CRYBA4-23 expression quantity is that real-time fluorescence determines PCR detection reagents in the detection Pancreatic Adenocarcinoma
Box.
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-CRYBA4-23 expression is included, primer sequence is SEQ NO:2,3 and reference gene
The primer sequence of TUBA1A 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-CRYBA4-23 from pancreas
RNA is extracted in cancerous tissue and carries out all reagents of reverse transcription and quantitative fluorescent PCR.Including：
(1) the extracted total RNA agents useful for same from the tumour of cancer of pancreas 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-CRYBA4-23 reverse transcriptions by template of total serum IgE, including reverse transcription reaction buffer solution,
Containing containing Mg2+Triphosphoric acid base deoxynucleotide dNTP, reverse transcriptase M-MLV and random primer and Oligo dT primers
Mixture；
(3) by cDNA real-time quantitative PCR agents useful for same, including Lnc-CRYBA4-23 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-CRYBA4-23 in Pancreatic Adenocarcinoma source by quantitative fluorescent PCR and survivorship curve analysis
Related to the survival rate of patient, content is higher, and survival rate is higher.This method is to predict that the prognosis life cycle analysis of Pancreas cancer patients carries
Strong technical support has been supplied, the postoperative life quality of Pancreas cancer patients is helped to improve, has worked out aftertreatment scheme, has been improved
Survival rate has far-reaching clinical meaning.And through site test, achieve very satisfied advantageous effects, relevant information
It is as follows：
The reagent of the preparation detection Lnc-CRYBA4-23 expression quantity of embodiment 1 is used to prepare the kit of Pancreas cancer patients prognosis
(being used for 30 secondary responses)
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. triphosphoric acid base deoxynucleotide dNTP 100 μ ls of the 10mM containing Mg2+
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-CRYBA4-23 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-CRYBA4-23 of embodiment
1, cancer of pancreas to be measured or normal control tissue are collected, after physiological saline cleans up, is put into and fills inhibition RNA degradation solvents
Cryopreservation tube in, 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-CRYBA4-23 RNA：It is tried using the reverse transcription of the green skies Bioisystech Co., Ltd in Shanghai
Agent box（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-CRYBA4-23：Specific primer is in raw work biology work
Journey（Shanghai）Limited liability company synthesizes, and includes the specific primer for detecting Lnc-CRYBA4-23 expression, and primer sequence is
SEQ NO:2, the primer sequence that 3 and reference gene TUBA1A is expressed, primer sequence are SEQ NO: 4、5.Real-time quantitative
The other reagents of PCR utilize the BeyoFast SYBR Green qPCR Mix (2 of the green skies Bioisystech Co., Ltd in Shanghai
×), it is as follows.
（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-CRYBA4-23 expression quantity：This experimental data is included in 60 Pancreas cancer patients and its normal
Control tissue.The interpretation of result of real-time quantitative PCR uses relative quantitation method, that is, 2^-△△CtMethod.It is specific as follows：It first, will be primary
All gene C t values of experiment are put in order, later with the Ct values of the target gene Lnc-CRYBA4-23 in each group of tumor sample
The Ct values of itself reference gene TUBA1A are subtracted, obtained number is exactly △ Ct；Changing formula into is exactly：△Ct=Ct（Target gene
Lnc-CRYBA4-23）-Ct（Reference gene TUBA1A）；Then, by each group of each target gene of tumor sample Lnc-
The △ Ct of CRYBA4-23 are calculated.Normal control tissue group sample is subtracted with the △ Ct of tumor tissues sample in this experiment
△ Ct, and opposite number is taken to all results simultaneously, the result which obtains is exactly-△ △ Ct.Finally, p- △ △ Ct into
The power operation of row 2, i.e. 2^-△△CtJust show that the multiple of expression quantity changes.In triplicate, it is counted using nonparametric t- inspections
Analysis.It was found that the expression quantity of Lnc-CRYBA4-23 is higher than normal control in the tumor tissues of cancer of pancreas（See Fig. 1）, difference
With significant difference (p＜ 0.05).
6, by 60 Pancreas cancer patients follow-up statistics being included in above-mentioned experiment, including patient's First episode
Time, treatment, recurrence status and death time etc., follow up time are at least 12 months.In selected Pancreas cancer patients
In, the expression value for choosing fluorescence real-time quantitative PCR analysis is reference standard, and the corresponding normal structure of acquired results is compared
Compared with.Lnc-CRYBA4-23 expression is defined as Lnc-CRYBA4-23 high expression higher than the patient of normal control tissue in tumor tissues
Group, remaining is low expression group.By Kaplan-Meier survival analysis, Lnc-CRYBA4-23 high expresses the life cycle ratio of patient
The patient of Lnc-CRYBA4-23 low expression groups is substantially reduced, and prognosis is worse（See Fig. 2）, difference have it is statistically significant (p＜
0.05).Therefore, Lnc-CRYBA4-23 can be used as the specificity molecular marker of Pancreas cancer patients prognosis.
By above it should be apparent that the invention discloses a kind of new cancer of pancreas prognosis molecule marker non-codings
RNA Lnc-CRYBA4-23 are used to prepare the kit of Pancreas cancer patients, the Lnc- tissue-derived by detecting pancreatic tumour
The kit of CRYBA4-23.Lnc-CIT-1 up-regulated expressions in cancer of pancreas, height expression Lnc-CRYBA4-23 are confirmed by research
Pancreas cancer patients, overall survival is worse.Therefore, by detecting Lnc-CRYBA4-23 in Pancreas cancer patients tumor tissues
Expression, Pancreas cancer patients can be made prognosis inspection diagnosis, accuracy rate is up to 96% or more, and is treated in time,
Survival and life quality are improved, there is far-reaching clinical application significance and application value, economic and social benefit
It is huge.
Sequence table
<110>The first affiliated hospital of Zhengzhou University
<120>A kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc-CRYBA4-23 and its application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 962
<212> DNA
<213> Homo sapiens
<400> 1
caggctggag tagacaggaa ccacgagagg cagggaggac tgtacagggg ttgctgacca 60
ggtggaagat gatgggagcc tcagaaggtg tcgtgggaca gggtcaggga gagggctcta 120
tgcggccagg atctggatgc agattgttga gactgaaatt tggctcagat cccttctagt 180
tgtgtctgtt tatcttcagt ttccttgtct ataaaatggg gagagattgt gtgtccccgg 240
catcactgga ctgttaatag ctattgcatt tatagcattt atcccagaac ctgaaacaca 300
gtaaatgctc agtaagtgtt attatttgcc ccaaactgtc ttagtgcttt ccccaagagc 360
tttgtgttgt ttgtactatg aggaataatg agggtggtgt gggagtcggc ctctgtgtct 420
gcagagagga ccacaggcca caggaataga gacaggtaga taggatcaag gtgacaggaa 480
cagatgttca gagcaagaga tgaagacggc atcatcaacc cagacaagcc agttggaggc 540
atctgtccac ccatgtggtt ccagacacgt tcatgtggcc accatgaccc cgtcggcatc 600
acaggggtaa ccaagagagc agcaagttcc tgggacagag gagacagaag ggaatttcca 660
ggttcttcag gacgttcaca accacactga gaagcatctt tgcagataag tatttaaact 720
taccagccca tagcctacca gcctgccctc ttctggtctg tgcaggaaag tgtagtatcc 780
tagaatgcca aagtgggagg ggaaatgggt gatgtagctc attctctttt ctacctctat 840
ggaagaaaga aagagcctgt ccccattttg tggggtccca gaaagggtga ttttcacact 900
tcacatttgg cgttagggct agtatttcac aaacattacc gtctggaact tttgaaggct 960
ga 962
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 2
gagaggcagg gaggactgta 20
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 3
ttggttaccc ctgtgatgcc 20
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 4
actggcttca aggttggcat 20
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 5
agtgggaggc tggtagttga 20
Claims (3)
1. a kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc-CRYBA4-23 is long-chain non-coding RNA, transcription
This length is more than 200 nucleotide, which is classified as SEQ No：1.
2. cancer of pancreas prognosis molecule marker non-coding RNA Lnc-CRYBA4-23 described in claim 1 is preparing cancer of pancreas
Application in prognosis real-time fluorescence quantitative PCR detection kit.
3. the cancer of pancreas prognosis molecule marker non-coding RNA Lnc-CRYBA4-23 described in claim 2 is preparing cancer of pancreas
Application in prognosis real-time fluorescence quantitative PCR detection kit, which is characterized in that the cancer of pancreas prognosis real time fluorescent quantitative
PCR detection kit includes the specific primer for the kit expression for detecting Pancreas cancer patients prognosis being placed in box body,
Primer sequence is SEQ No：2 and 3, the primer sequence of reference gene TUBA1A expression, primer sequence are SEQ No：4,5 and from pancreas
RNA is extracted in glandular tissue, and carries out the reagent of reverse transcription and quantitative fluorescent PCR.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111118154A (en) * | 2020-01-16 | 2020-05-08 | 华南协同创新研究院 | Application of LINC01272 in preparation of tumor detection reagent and/or treatment drug |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017137427A1 (en) * | 2016-02-08 | 2017-08-17 | Ucl Business Plc | Detection of cancer of the esophagus |
| CN107727865A (en) * | 2016-08-11 | 2018-02-23 | 博尔诚（北京）科技有限公司 | The systemic detection method of tumor markers and its application |
-
2018
- 2018-04-24 CN CN201810373567.1A patent/CN108424965A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017137427A1 (en) * | 2016-02-08 | 2017-08-17 | Ucl Business Plc | Detection of cancer of the esophagus |
| CN107727865A (en) * | 2016-08-11 | 2018-02-23 | 博尔诚（北京）科技有限公司 | The systemic detection method of tumor markers and its application |
Non-Patent Citations (4)
| Title |
|---|
| ENSEMBL: ""Transcript: MIAT-005 OTTHUMT00000320800.1"", 《VEGA GENOME BROWSER》 * |
| MENG ZHOU ET AL.: ""Construction and analysis of dysregulated lncRNA-associated ceRNA network identifed novel lncRNA biomarkers for early diagnosis of human pancreatic cancer"", 《ONCOTARGET》 * |
| SOREN MULLER ET AL.: ""Next-generation sequencing reveals novel differentially regulated mRNAs, lncRNAs, miRNAs,sdRNAs and a piRNA in pancreatic cancer"", 《MOLECULAR CANCER》 * |
| TING-FU LI ET AL.: ""The interaction of long non-coding RNA MIAT and miR-133 play a role in the proliferation and metastasis of pancreatic carcinoma"", 《BIOMEDICINE & PHARMACOTHERAPY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111118154A (en) * | 2020-01-16 | 2020-05-08 | 华南协同创新研究院 | Application of LINC01272 in preparation of tumor detection reagent and/or treatment drug |
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Application publication date: 20180821 |