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Colorectal cancer microRNA molecular marker and application thereof

A molecular marker, colorectal cancer technology, applied in DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problem of less differential expression research

Active Publication Date: 2018-03-09
北京旷博生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In recent years, there have been many studies on miRNA markers of colorectal cancer, but they mainly focus on the differential expression of miRNA between colorectal cancer tissue and normal colorectal tissue, and there are few studies on the differential expression of serum / plasma miRNA. Find microRNA markers that can effectively judge colorectal cancer, especially microRNA markers that can be used to distinguish colorectal cancer from benign diseases such as polyps

Method used

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  • Colorectal cancer microRNA molecular marker and application thereof
  • Colorectal cancer microRNA molecular marker and application thereof
  • Colorectal cancer microRNA molecular marker and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Serum sample collection and preparation

[0044] Between January 2013 and January 2016, 100 serum samples from patients meeting the above definition of colorectal cancer, 100 serum samples from patients meeting the above definition of colorectal polyps, and 100 serum samples from healthy controls were pre-selected from 301 hospital collection.

[0045] Collect 6ml of peripheral venous blood without anticoagulation. The serum collection process is as follows: put the whole blood sample at room temperature for 2 hours to allow it to coagulate fully; place it in a centrifuge at 4°C and centrifuge at 3,500rpm for 7 minutes. Carefully transfer the upper plasma layer to a 1.5 mL RNase-free sterile centrifuge tube with a 200 μL pipette. Label each sample. Be sure to store the serum sample in an ultra-low temperature (-80°C) refrigerator within 12 hours.

Embodiment 2

[0046] Example 2. Extraction of total RNA in serum

[0047] Use the RNA extraction kit (Beijing Kuangbo Biotechnology Co., Ltd.) to extract total RNA from serum, and add 1 μl (20 nM) of ExternalControl-1 (Shanghai Shengsheng) with the sequence 5'-CAACCTCCTAGAAAGAGTA-3' (EC1) to every 250 μl of serum. Synthesized by Gong Biological Engineering Technology Co., Ltd.) to monitor the extraction quality of RNA in serum. The concentration of extracted total RNA was determined using Thermo NanoDrop 2000c.

Embodiment 3

[0048] Example 3. Three-step method for quantitative detection of microRNA in serum

[0049] (1) Add polyA tail:

[0050] i. Prepare a polyA-tailed reaction solution in an RNase-free PCR tube (Axygen, 200 μl), with a volume of 20 μl. Add 1 μl (20nM) of ExternalControl-2 (synthesized by Shanghai Sangon Biotechnology Co., Ltd.) with the sequence 5’-TGAGCAACGCGAACAA-3’ (EC2) to each 20 μl system to monitor the quality of microRNA tailing and reverse transcription.

[0051]

[0052] (Note: The enzymes used in this experiment are all products of Beijing Kuangbo Biotechnology Co., Ltd.)

[0053] ii. Put the PCR tube containing the prepared reaction solution into a PCR machine (Thermo) and incubate at 37° C. for 1 hour. (2) RT-PCR to obtain cDNA single strand:

[0054] i. Add 0.5 μl (0.5ng / μl) to the reaction solution obtained in (1) RT-Primer (synthesized by Shanghai Sangong Bioengineering Technology Co., Ltd.) whose sequence is 5'-CAGTGGTATCAACGCACTCCTTTTTTTTTTTTTTTTTTTTTTTTT...

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Abstract

The invention provides a colorectal cancer microRNA molecular marker combination which comprises more than one microRNA nucleic acid molecule selected from: miR-21-3p, miR-877-5p, miR-181d, miR-202-3p, miR-19b-3p, miR-380-5p, miR-1246, miR-627, miR-451a, miR-339-5p, miR-520e, miR-21-5p and miR-532-3p, as well as application to preparation of a colorectal cancer diagnostic kit, and the colorectal cancer diagnostic kit. The comparison between the content of microRNA molecular markers of a colorectal cancer patient in plasma / serum and the content of microRNA molecular markers of colorectal polyppatient and health comparison in plasma / serum shows a great difference. The molecular marker used for guiding diagnoses has the characteristics of high sensitivity, high specificity and the like.

Description

technical field [0001] The present invention relates to the field of diagnosis of colorectal cancer, and specifically relates to a set of colorectal cancer microRNA molecular marker combinations, which can be applied to the diagnosis, curative effect and prognosis evaluation of colorectal cancer. Through the diagnosis of colorectal cancer, timely detection and treatment of colorectal cancer is very helpful for the control and cure of colorectal cancer. For patients with colorectal cancer, increasing the accuracy of diagnosis can determine the targeted prevention and treatment plan, and evaluate the curative effect and prognosis, greatly improving their clinical cure rate and long-term survival rate. It can be applied to the early diagnosis of suspected tumor patients, as well as the treatment and tracking of confirmed patients. Background technique [0002] microRNA (miRNA) is a research hotspot in recent years. It is a single-stranded small molecule RNA widely present in e...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/113
CPCC12Q1/6886C12Q2600/178C12Q2600/118C12Q2600/158
Inventor 贾宝庆刘洪一张卫红李冰靳文静李鹏宋高尚郭少华刘欣欣
Owner 北京旷博生物技术股份有限公司
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