CN108546760A - A kind of cancer of pancreas prognosis molecule marker non-coding RNA Lnc-COL20A1-2 and its application - Google Patents

Abstract

The present invention relates to cancer of pancreas prognosis molecule marker non-coding RNA Lnc COL20A1 2 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 COL20A1 2, its transcript length is more than 200 nucleotide, and sequence is SEQ NO:1；Application in preparing cancer of pancreas prognosis real-time fluorescence quantitative PCR detection kit, the kit include 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 COL20A1 2 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

A pancreatic cancer prognostic molecular marker non-coding RNA Lnc-COL20A1-2 and its application

technical field

The invention relates to tumor molecular biology, in particular to a pancreatic cancer prognosis molecular marker non-coding RNA Lnc-COL20A1-2 and its application.

Background technique

Pancreatic cancer is one of the most malignant tumors, with insidious onset, rapid progression, low surgical resection rate, poor effect of radiotherapy and chemotherapy, and extremely poor prognosis. It ranks 13th and ranks fourth among all causes of death from malignant tumors. Most patients with pancreatic cancer often have lymphatic or blood supply metastasis when they are diagnosed, thus losing the opportunity for radical surgery. The main cause of death in patients with pancreatic cancer is local invasion or distant metastasis in the early stage of the tumor, which is also the main factor restricting the prognosis of patients. At present, the mechanism of invasion and metastasis of pancreatic cancer is still unclear. Understanding the mechanism of malignant biological behavior of pancreatic cancer and giving appropriate intervention measures are of great practical significance for improving the clinical diagnosis and treatment of pancreatic cancer and improving the prognosis of patients. At present, there is no ideal molecular index to evaluate the prognosis and the risk of metastasis clinically. Therefore, finding new prognostic biomarkers is an urgent problem to be solved.

In recent years, the development of next-generation sequencing technology has greatly promoted the progress of biology. After the Human Genome Project, the genomes of a large number of species were sequenced. The annotation of these genomes is of great concern to researchers. The central dogma has greatly promoted the development of modern molecular biology. RNA, as a bridge for the transmission of genetic information between DNA and protein, has always been highly concerned by scientists. With the development of science, RNAs of various types and functions have been discovered. The function of RNA has gone far beyond the traditional understanding as an intermediary of genetic information transmission. Many types of RNA molecules (such as microRNAs, long-chain non-coding RNAs, etc.) can directly function. It has been found that more than 80% of the human genome can be transcribed into RNA, while only about 2% of the region can be translated into protein, and most of the transcripts are non-coding RNA, which has aroused the interest of many researchers. How to effectively identify these non-coding RNAs and determine their functions is a major challenge in current research work. More and more studies have shown that non-coding RNAs are not "garbage" produced by transcription, but play an important role in transcription and translation regulation. Lnc-COL20A1-2 is a newly discovered non-coding RNA with a transcript length greater than 200 nucleotides. At present, the important role of non-coding RNA Lnc-COL20A1-2 in the process of normal cell development or tumor development is still under further study. Whether Lnc-COL20A1-2 can be used as a new tumor marker to predict the prognosis of pancreatic cancer patients has not been reported yet.

Contents of the invention

In view of the above situation, in order to overcome the defects of the prior art, the purpose of the present invention is to provide a pancreatic cancer prognosis molecular marker non-coding RNA Lnc-COL20A1-2 and its application, which can effectively solve the problem of preparing a kit for predicting the prognosis of pancreatic cancer patients. The problem.

The technical solution solved by the present invention is a pancreatic cancer prognosis molecular marker non-coding RNA Lnc-COL20A1-2, the transcript length of which is greater than 200 nucleotides, and the sequence is SEQ NO:1;

Application of the pancreatic cancer prognosis molecular marker non-coding RNA Lnc-COL20A1-2 in the preparation of a pancreatic cancer prognosis real-time fluorescent quantitative PCR detection kit;

The pancreatic cancer prognosis real-time fluorescent quantitative PCR detection kit includes specific primers expressed in the kit for detecting the prognosis of pancreatic cancer patients placed in the box, and the primer sequences are SEQ No: 2 and 3, and the expression of the internal reference gene TUBA1A The primer sequence, the primer sequence is SEQ No: 4, 5 and the reagents for extracting RNA from pancreatic cancer tissue, performing reverse transcription and fluorescent quantitative PCR.

Lnc-COL20A1-2 of the present invention is a newly discovered non-coding RNA, which can be effectively used to prepare a kit for the prognosis of patients with pancreatic cancer. As a new molecular marker for the prognosis of pancreatic cancer, the non-coding RNA Lnc-COL20A1-2 can be used in time Predicting the prognosis of pancreatic cancer and treating pancreatic cancer patients in time to prolong the survival time of patients and improve the survival rate is a major innovation in the prognosis of pancreatic cancer, with huge economic and social benefits.

Description of drawings

Fig. 1 is a graph showing the difference in expression of Lnc-COL20A1-2 in normal tissues and pancreatic cancer analyzed by real-time fluorescent quantitative PCR of the present invention.

Fig. 2 is a graph showing the influence of the present invention on the prognosis of pancreatic cancer patients and the survival curve analysis of Lnc-COL20A1-2 expression levels derived from pancreatic cancer tissues.

Detailed ways

The specific implementation of the present invention will be described in detail below in combination with specific situations.

In the specific implementation of the present invention, a pancreatic cancer prognosis molecular marker non-coding RNA Lnc-COL20A1-2 is a newly discovered non-coding RNA Lnc-COL20A1-2 molecular marker, which is a long-chain non-coding RNA. The length of the transcript is greater than 200 nucleotides, and the nucleotide sequence is SEQ No: 1:

SEQ NO:1

GTAGGTAAAGAAAGGCAGATTTATTAGAGAAAGTAGAAAATGTGCATAGCACAGAAGCAACGGGCAGGTCAGCAAGGGAGGAGCCGAAGCAAGGGGCAGGTCAGCAAGGGAGGAGCCGAAGCAAGGGGCAGGTCAGCAAGGGAGGAGCCGAAGCAAGGGGCAGGTCAGCAAGGGAGGAGCCGAAGCAAGGGGCAGGTCAGCAAGGGAGGAGCCACTGCAAAGAAATGAGGGCTTGCTGGGGATGTTATAGGACGGCGCCTGTGCTGTGTGCTGGAGAGGGCCACAGGCAGTACTGATAACGCCAAGGTTGCAGTCAGCTAACATCCATTCTCCTATCAGCCAAGGGTCTGGCAATAGCCGGGCACAGGAAGATTGTGAGTTATTTCTTCAGGAGGGCTGTGTGTCCTGGACAGTGAAGAAGGGCAGACTTAAAGCTTCTTTGCTTCTTCTTTTTGCCTTCCCTCTGTCCTGCCAGCCCGACTCCTTTTCCCTCATTAGGATTCCACACCCCCCACCCCCACATGTCAGGGCACAGCCGGGCTGACCAGCTCCACCCGCTCTGCTGGGCCCTCACTGAGTCTCAGTCTTCTCATGTGTGACACAGGCAAGGTGAACATGGTGGACATGTGTCAGGCGGGCGCCTGGGCCTAGAGGAGGAAGGAGCAGGGCCATTGTGGGGGCGCAGGCTGGGCCTGGCTCCTGGGGCCTGGGCCCAGCTCCTGGGGCCTGGGCCCAGAGGAGGAAGGAGCAGGGCCGGTGTGGGGGCGCAGGCTGGGCCTGGCTCCTGGGGCCTGGACCTAGAGGAGGAAGGAGCAGGGCCGGTGTGGGGGCGCAGGCTGGGCCTGGCTCCTGGGGCCTGGGCCCAGAGGAGGAAGGAGCAGGGCCGGTGTGGGGGCGCAGGCTGGGCCTGGCTCCTGGGGCCTGGACCTAGAGGAGGAAGGAGCAGGGCCGGTGTGGAGGTGCAGGCTGGGCCTGGCTCCTGGGGCAATAATTGCTGTGG GCGGGGAGTGCCAGGGCTGGGCCATGGCCCCCTAGGCTCTCTGTGCACAGCCCCCCAGTAGGCCCGTGGGACTTGCAGGCTCTCAGCAAATCTCCCGTCAGAAGCTGGTGGCTGCAGGTTTGACACCCCGTCGTCCCATGGAGTTAGCGAGCACCCATTCTGCACCTGCTGTGTACGCGGCAGGATCCGGGAGGCAGTGCCAGCCCTCAGGGAGGACGGAAGGGTGGATGGATGCGTGGTGGCTGCTCCCGGCTGCAGGGAGCATACAGCACAGGCGGACTGGACACCAGCTGAATAGCCTGTCCTGCGCTGTGCTGGGGTGCAAGGGAATGAGCCACGTCAGCCCCGACCCAAGTGGAGCTGACCAGGGCTGCGTCCATGAAATGATGGAGGAGCCAGGTGTGGTCAGAATTTGCCAGGGAACAGTGACCCTCCAAGAGGGCCGCTCAGGGAAGCTTCATAGAGGAAGCAGCGCTTGGGCGAGGACTTGGGGTTGGGGGAGGGCCAGGAAGGGGAGCTGCGTCCTTCCAAGACAAGGGGGCTGGAGGGCTCGGTGGGAGCAGAGGCGGGGCCGCTCCATGAGCTACACAGCACCAAGTCTGCGCCTGGGTGGTCTCAGGTCCCAGAGCCTCCCTCTGGCAGGGCAGGCAATGAGGCAGGTGGCCCATCCGGGGATCACTGCCTGAGCAACCAGCAGGATTTGGTCTCCGGCCAGCAGCAGGGAGGAAGTTAACATAAATCCATTCCAATGCAAATGTCAAACCAACCGTGCGCAGGAAAACTTAGCAGCTCTGAATGTCAAGGAGGGAGTCCCGGCTCTAGCTGGTGGCGCGGCTTGGCTGAGCTGTTTCCTCCAGCTCCTGGGCTGTGGGAGGAGGCTGCTGGCGTGCGGCCACTGCCTGGCGCAGAGGGGCTGCCAGGACCTTTGAGAACTGAGGCACACCCGGGAGATGATCTGGGGACTCGTCGCTGGAGGGAAGGAGCCCCAGCAAACACAGCC CAGTCCAGGGACTCTTGTCTGCATAGCCTCTGAGACACGCGGGAGCTGGTGCCCGGGGTCCTGAGTCCAGGCGGGGGCTCCACCCTGAGGCGTGCGTGGGTGACGGGCTCCCAAGACATGACCACGCTGGGTGTGAAGACAAGGCCACGTGAGGCCGGAGGACAGTGCAG 2170

Application of the pancreatic cancer prognosis molecular marker non-coding RNA Lnc-COL20A1-2 in the preparation of a pancreatic cancer prognosis real-time fluorescent quantitative PCR detection kit;

The pancreatic cancer prognosis real-time fluorescent quantitative PCR detection kit includes specific primers expressed in the kit for detecting the prognosis of pancreatic cancer patients placed in the box, and the primer sequences are SEQ No: 2 and 3, and the expression of the internal reference gene TUBA1A The primer sequence, the primer sequence is SEQ No: 4, 5 and the reagents for extracting RNA from pancreatic cancer tissue and performing reverse transcription and fluorescent quantitative PCR, wherein:

SEQ NO:2

GTCCTTCCAAGACAAGGGGG

SEQ NO:3

GCAGACAAGAGTCCCTGGAC

SEQ NO:4

aaaggggtggggggagagatt

SEQ NO:5

gttccaggcagtagagctcc

The kit for detecting the expression level of Lnc-COL20A1-2 in pancreatic cancer tissue is a real-time fluorescent PCR detection kit.

The real-time fluorescent quantitative PCR detection kit includes specific primers for real-time fluorescent quantitative PCR, including specific primers for detecting the expression of Lnc-COL20A1-2, the primer sequence is SEQ NO: 2, 3, and the internal reference gene TUBA1A The expressed primer sequence is SEQ NO: 4,5.

The real-time fluorescent quantitative PCR detection kit, in addition to the primers of Lnc-COL20A1-2, also contains all reagents for extracting RNA from pancreatic cancer tissue and performing reverse transcription and fluorescent quantitative PCR. include:

(1) Reagents for extracting total RNA from pancreatic cancer tumors or normal control tissues, including Trizol solution, chloroform, isopropanol, RNA degradation inhibiting solvent, 75% ethanol, and DEPC water;

(2) Reagents for reverse transcription of Lnc-COL20A1-2 into cDNA using total RNA as a template, including reverse transcription reaction buffer, containing Mg2 ⁺ -containing triphosphate base deoxynucleotide dNTP, reverse transcriptase M-MLV and a mixture of random primers and Oligo dT primers;

(3) Reagents used for cDNA real-time quantitative PCR, including Lnc-COL20A1-2 real-time fluorescent quantitative PCR specific primers, TUBA1A internal reference specific PCR primers, real-time fluorescent quantitative SYBR dyes, and RNase-free water.

The present invention finds that the Lnc-COL20A1-2 derived from pancreatic cancer tissue is related to the survival rate of patients through fluorescent quantitative PCR and survival curve analysis, and the higher the content, the higher the survival rate. This method provides strong technical support for predicting the prognosis and survival of pancreatic cancer patients, and is helpful to improve the postoperative life quality of pancreatic cancer patients, formulate postoperative treatment plans, and improve survival rate, which has far-reaching clinical significance. And through field tests, very satisfactory beneficial technical effects have been obtained. The relevant information is as follows:

Example 1

Preparation of reagents for detecting the expression of Lnc-COL20A1-2 is used to prepare a kit for the prognosis of patients with pancreatic cancer (for 30 reactions)

1. Trizo l20ml

2. Inhibit RNA degradation solvent 40ml

3. Chloroform 80ml

4. Isopropanol 80ml

5. DEPC water 10ml

6. Mixture of 10×random primers and Oligo dT primers 100μl

7. 5× Reverse Transcription Reaction Buffer 150μl

8. 10mM deoxyribonucleotide triphosphate base dNTP containing Mg ²⁺ 100μl

9. 200U/μl M-MLV reverse transcriptase 50μl

10. SYBR Green qPCR Mix 500μl

11. 3μM target gene Lnc-COL20A1-2 specific primer (its sequence is shown in SEQ NO: 2, 3) 50μl

12. 3μM internal reference gene TUBA1A-specific primer (its sequence is shown in SEQ NO: 4, 5) 50μl

Example 2 Detection of Lnc-COL20A1-2 in tissue samples

1. Collect the pancreatic cancer or normal control tissues to be tested, wash them with normal saline, put them into a cryopreservation tube filled with a solvent that inhibits RNA degradation, and put them in a -80°C refrigerator for later use.

2. Extraction of RNA in tissues:

(1) Add liquid nitrogen to the mortar first, then cut the tissue into small pieces and grind it into powder in liquid nitrogen, take 100mg of tissue powder with a liquid nitrogen pre-cooled spoon and add it to the EP tube filled with 1ml of Trizol solution middle. The total volume of tissue powder should not exceed 10% of the volume of Trizol used, and it should be thoroughly mixed evenly.

(2) Leave at room temperature for 5 minutes, then add 200 μL of chloroform, tightly cap the EP tube and shake vigorously for 0.5 minutes. Centrifuge at 12,000 rpm for 10 minutes.

(3) Take the upper aqueous phase into a new EP tube (do not mix the middle precipitated layer and the lower layer), add 500 μL of isopropanol, and gently invert to mix. Place at room temperature for 10 minutes, and centrifuge at 12,000 rpm for 10 minutes.

(4) Carefully discard the supernatant, add 1ml of 75% ethanol, vortex, and centrifuge at 12000 rpm for 5 minutes at 4°C. Repeat the operation once.

(5) Discard the supernatant (remove the residual liquid as much as possible), and dry at room temperature or in vacuum for 5-10 minutes (be careful not to dry too much, otherwise the solubility of RNA will be reduced). Dissolve the RNA with 50 μL of DEPC-treated water.

(6) Measurement of RNA concentration: measure with a nucleic acid concentration measuring instrument, add 1 μL of RNA sample to the sample hole, and directly determine the concentration of RNA according to the reading. The OD260/OD280 ratio measured by the nucleic acid concentration analyzer, if the ratio is between 1.8-2.0, the RNA purity is considered to be very good. Finally, the RNA was stored in a -80°C refrigerator for future use.

3. Reverse transcription of Lnc-COL20A1-2 RNA: use the reverse transcription kit (D7170S) of Shanghai Beyontien Biotechnology Co., Ltd. Specific steps are as follows.

(1) Genomic DNA removal: thaw template Total RNA, 5×gDNA Eraser Buffer on ice, thaw 5×RT Buffer, 10×RT Primer Mix, DEPC-treated Water at room temperature (15-25°C), and thaw quickly Keep on ice. Mix each solution well and centrifuge briefly before use to allow all liquid to settle to the bottom of the tube. For the denaturation of RNA, heat denature the RNA sample at 65°C for 5 minutes, and immediately place it in ice water to cool. Prepare the reaction mixture on ice according to the components in the table below, then dispense into each reaction tube, and finally add the RNA sample.

Reagent Usage amount 5×gDNA Eraser Buffer 2μl Total RNA up to 0.5μg DEPC-treated Water to 10μl total capacity 10μl

(2) Incubate at 37°C for 2 minutes on a PCR machine or in a water bath. Quickly place on ice for later use.

(3) Reverse transcription system preparation: Prepare the reaction solution on ice, and perform reverse transcription reaction immediately after mixing gently. Prepare the mixed solution according to the reverse transcription reaction system in the table below.

Reagent Usage amount 5×RT Buffer (with Mg ²⁺ and dNTP) 4μl 10×RT Primer Mix (a mixture of Oligo dT Primer and Random Hexamers) 2μl BeyoRT™ II M-MLV Reverse Transcriptase (RNase H-) (with RNase Inhibitor) 2μl DEPC-treated water 2μl Total RNA after removal of gDNA above 10μl total capacity 20μl

(4) Incubate at 42°C for 60 minutes to carry out the reverse transcription reaction, then incubate at 80°C for 10 minutes to inactivate the reverse transcriptase and put it on ice. For templates with complex secondary structure or high GC content, the reverse transcription temperature can be increased to 50°C to enhance the efficiency of reverse transcription.

(5) The obtained cDNA can be used immediately or frozen at -80°C for subsequent real-time fluorescent quantitative PCR. It is advisable to avoid excessive repeated freezing and thawing of the cDNA.

4. Real-time quantitative PCR using specific primers of Lnc-COL20A1-2: specific primers were synthesized in Sangon Bioengineering (Shanghai) Co., Ltd., including specific primers and primer sequences for detecting the expression of Lnc-COL20A1-2 It is SEQ NO: 2, 3, and the primer sequence expressed by the internal reference gene TUBA1A, and the primer sequence is SEQ NO: 4, 5. Other real-time quantitative PCR reagents used BeyoFast™ SYBR Green qPCR Mix (2×) from Shanghai Beyotime Biotechnology Co., Ltd. The specific steps are as follows.

(1) Thaw and mix the various solutions required for the PCR reaction. BeyoFast™ SYBR Green qPCR Mix is completely thawed and mixed and placed in an ice box.

(2) Set up a PCR reaction system on an ice bath (taking a 96-well plate as an example):

Reagent Usage amount BeyoFast™ SYBR Green qPCR Mix (2X, Low ROX) 10μl Specific primers (concentration 3μM) 2μl template DNA 2μl RNase-free water 6μl total capacity 20μl

(3) Generally, the amount of DNA template is 1-10ng cDNA as a reference amount, and good detection effect can be obtained when the final concentration of primers is 0.2-0.5μM, and the final concentration of primers can also be adjusted according to the situation. If necessary, serially dilute the template to determine the optimal amount of template to use. When the cDNA obtained from the reverse transcription PCR reaction is directly used as a template, the amount added should not exceed 10% of the total volume of the PCR reaction. The recommended reaction system for a 96-well plate is 20 μl, and the reaction system can also be scaled up or down according to actual experimental needs.

(4) Gently pipette to mix or slightly Vortex to mix, and centrifuge at room temperature for a few seconds to make the liquid accumulate at the bottom of the tube.

(5) Place the set PCR reaction tube or PCR reaction plate on the fluorescent quantitative PCR instrument to start the PCR reaction.

(6) PCR reaction program: Pre-denature the template before the real-time fluorescent quantitative PCR reaction, usually set at 95°C for 2 minutes. The following PCR program is used. This program is based on the ABI 7900HT fluorescent quantitative PCR instrument as an example:

a. Pre-denaturation: 95°C for 2 minutes

b. Denaturation: 95°C 15sec

c. Annealing/extension: 60℃ 15-30sec

d. Repeat steps b and c for a total of 40 cycles

e. Melting curve analysis (optional): 95°C 15sec, 60°C 15sec, 95°C 15sec

f. Use the software provided by the fluorescent quantitative PCR instrument to analyze the results

The three-step method only needs to add a step of 72°C for 30 sec after annealing/extension, and then repeat steps b, c and this additional step for a total of 40 cycles.

5. Data analysis of Lnc-COL20A1-2 expression level: The experimental data included 60 cases of pancreatic cancer patients and their normal control tissues. The results of real-time quantitative PCR were analyzed by relative quantitative method, that is, 2^ ^-△△Ct method. The details are as follows: First, sort out the Ct values of all genes in an experiment, and then subtract the Ct value of the internal reference gene TUBA1A from the Ct value of the target gene Lnc-COL20A1-2 in each group of tumor samples, and the obtained number is △ Ct; replaced by the formula: △Ct=Ct (target gene Lnc-COL20A1-2)-Ct (internal reference gene TUBA1A); then, calculate the △Ct of each target gene Lnc-COL20A1-2 in each group of tumor samples it is good. Subtract the △Ct of the normal control tissue group from the △Ct of the tumor tissue sample in this experiment, and take the inverse of all the results at the same time. The result of this step is -△△Ct. Finally, perform a power of 2 operation on -△△Ct, that is, 2^ ^-△△Ct to get the fold change of the expression amount. Statistical analysis was performed using a nonparametric t-test in triplicate. The expression of Lnc-COL20A1-2 in pancreatic cancer tumor tissue was higher than that in normal control (see Figure 1), and the difference was statistically significant ( p <0.05).

6. Through the follow-up statistical data of 60 pancreatic cancer patients included in the above experiment, including the time of first onset, treatment, recurrence and death time of the patients, the follow-up time is at least 12 months. In the selected patients with pancreatic cancer, the expression value of the fluorescent real-time quantitative PCR analysis was selected as a reference standard, and the obtained results were compared with the corresponding normal tissues. Patients whose tumor tissue expressed Lnc-COL20A1-2 higher than normal control tissue were defined as the Lnc-COL20A1-2 high expression group, and the rest were low expression group. Through Kaplan-Meier survival analysis, the survival period of patients with high expression of Lnc-COL20A1-2 was significantly shorter than that of patients with low expression of Lnc-COL20A1-2, and the prognosis was worse (see Figure 2), and the difference was statistically significant ( p < 0.05). Therefore, Lnc-COL20A1-2 can be used as a specific molecular marker for the prognosis of pancreatic cancer patients.

It can be clearly seen from the above that the present invention discloses a new pancreatic cancer prognosis molecular marker non-coding RNA Lnc-COL20A1-2, which is used to prepare a kit for patients with pancreatic cancer. By detecting the Lnc derived from pancreatic cancer tumor tissue - Kit for COL20A1-2. Studies have confirmed that the expression of Lnc-CIT-1 is up-regulated in pancreatic cancer, and the overall survival rate of pancreatic cancer patients with high expression of Lnc-COL20A1-2 is worse. Therefore, by detecting the expression level of Lnc-COL20A1-2 in the tumor tissue of pancreatic cancer patients, the prognosis of pancreatic cancer patients can be diagnosed with an accuracy rate of over 96%, and timely treatment can improve the survival rate and quality of life of patients. It has far-reaching clinical application significance and popularization and application value, and has huge economic and social benefits.

sequence listing

<110> The First Affiliated Hospital of Zhengzhou University

<120> A pancreatic cancer prognostic molecular marker non-coding RNA Lnc-COL20A1-2 and its application

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ctcctttcc ctcattagga ttccacaccc cccaccccca catgtcaggg cacagccggg 540

ctgaccagct ccacccgctc tgctgggccc tcactgagtc tcagtcttct catgtgtgac 600

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gaaatgatgg aggagccagg tgtggtcaga atttgccagg gaacagtgac cctccaagag 1440

ggccgctcag ggaagcttca tagaggaagc agcgcttggg cgaggacttggggttggggg 1500

agggccagga aggggagctg cgtccttcca agacaagggg gctggagggc tcggtggggag 1560

cagaggcggg gccgctccat gagctacaca gcaccaagtc tgcgcctggg tggtctcagg 1620

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gcctgagcaa ccagcaggat ttggtctccg gccagcagca gggaggaagt taacataaat 1740

ccattccaat gcaaatgtca aaccaaccgt gcgcaggaaa acttagcagc tctgaatgtc 1800

aaggaggggag tcccggctct agctggtggc gcggcttggc tgagctgttt cctccagctc 1860

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Claims (3)

1. A non-coding RNA Lnc-COL20A1-2, a pancreatic cancer prognostic molecular marker, is a long-chain non-coding RNA whose transcript length is greater than 200 nucleotides. The nucleotide sequence is SEQ No:1. 2. The application of the pancreatic cancer prognosis molecular marker non-coding RNA Lnc-COL20A1-2 according to claim 1 in the preparation of a pancreatic cancer prognosis real-time fluorescent quantitative PCR detection kit. 3. The application of the non-coding RNA Lnc-COL20A1-2 of the pancreatic cancer prognosis molecular marker non-coding RNA Lnc-COL20A1-2 in the preparation of the pancreatic cancer prognosis real-time fluorescence quantitative PCR detection kit according to claim 2 is characterized in that, the described pancreatic cancer prognosis real-time fluorescence The quantitative PCR detection kit includes the specific primers expressed by the kit for detecting the prognosis of pancreatic cancer patients placed in the box, the primer sequences are SEQ No: 2 and 3, the primer sequence expressed by the internal reference gene TUBA1A, the primer sequence is SEQ No : 4, 5 and reagents for extracting RNA from pancreatic tissue, performing reverse transcription and fluorescent quantitative PCR.