microRNA molecular marker of liver cirrhosis and use thereof

A molecular marker and application technology, applied in DNA/RNA fragmentation, recombinant DNA technology, microbial determination/examination, etc., can solve the problem of lack of in-depth research on microRNA expression changes in patients with liver cirrhosis.

Inactive Publication Date: 2014-12-24
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

Although some studies have been carried out in this field, the changes of microRNA expression in patients with liver cirrhosis have not been studied in depth. Differentiating microRNA markers for hepatitis B

Method used

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  • microRNA molecular marker of liver cirrhosis and use thereof
  • microRNA molecular marker of liver cirrhosis and use thereof
  • microRNA molecular marker of liver cirrhosis and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Collection and preparation of plasma samples

[0042] Between June 2012 and March 2014, plasma samples from 150 patients meeting the above definition of cirrhosis and 150 plasma samples meeting the above definition of hepatitis B were pre-collected from Beijing Youan Hospital affiliated to Capital Medical University.

[0043] Collect 10ml of peripheral venous blood and anticoagulate with EDTA. The plasma collection process is as follows: put the whole blood sample in a centrifuge at 4°C and centrifuge at 1,500-3,000g for 15 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 plasma sample in an ultra-low temperature (-80°C) refrigerator within 4 hours.

Embodiment 2

[0044] Example 2. Extraction of total RNA in plasma

[0045] Use the RNA extraction kit (Beijing Kuangbo Biotechnology Co., Ltd.) to extract total RNA from the plasma, and add 1 μl (20 nM) of the sequence 5'-CAACCTCCTAGAAAGAGTA-3' (SEQ ID NO: 49) to each 250 μl of plasma. 1 (synthesized by Shanghai Sangon Bioengineering Technology Co., Ltd.) to monitor the extraction quality of RNA in plasma. The concentration of extracted total RNA was measured using Thermo NanoDrop2000c.

Embodiment 3

[0046] Example 3. Three-step method for quantitative detection of microRNA in plasma

[0047] (1) Add polyA tail:

[0048] 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 External Control-2 (synthesized by Shanghai Sangon Bioengineering Technology Co., Ltd.) with the sequence 5'-TGAGCAACGCGAACAA-3' (SEQ ID NO: 50) to each 20 μl system to monitor the tailing and inversion of microRNA recording quality.

[0049]

[0050] (Note: The enzymes used in this experiment are all products of Beijing Kuangbo Biotechnology Co., Ltd.)

[0051] 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:

[0052] i. Add 0.5 μl (0.5ng / μl) of RT-Primer (synthesized by Shanghai Sangon Bioengineering Technology Co., Ltd.) to the reaction solution obtained in (1) with the sequence 5'-CAGTGGTATCAACGCACTCC...

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Abstract

The invention provides microRNA molecular marker combination of liver cirrhosis. The combination comprises more than one microRNA molecules of nucleic acid with sequence numbers of 1-24 and 54. The invention further provides use of preparing a liver cirrhosis diagnostic reagent. Compared with the content of the microRNA molecular marker of a hepatitis B patient in plasma/serum, the content of the microRNA molecular marker of a liver cirrhosis patient in plasma/serum is remarkably different, so that the liver cirrhosis patient and the hepatitis B patient can be effectively differentiated. When the expression level of the microRNA molecular marker reaches the level of liver cirrhosis, liver cirrhosis can be diagnosed. The invention further provides a diagnostic reagent kit for liver cirrhosis. The microRNA molecular marker of liver cirrhosis provided by the invention is used for guiding diagnosis of liver cirrhosis and has the characteristics of being simple in operation, safe, free from damage, high in specificity, high in sensitivity and easy to screen massively.

Description

technical field [0001] The present invention relates to the field of diagnosis of liver cirrhosis, in particular to a group of liver cirrhosis microRNA molecular marker combinations, which can be applied to the diagnosis of liver cirrhosis, through the diagnosis of liver cirrhosis, timely detection and treatment of liver cirrhosis, for liver cirrhosis Disease control is very helpful, and can be effectively turned over to avoid the development of liver cirrhosis to liver cancer. Background technique [0002] microRNA (miRNA) is a research hotspot in recent years. It is a single-stranded small molecule RNA widely present in eukaryotes. It has no coding function, but it can bind to the flanking region of the gene sequence to repress or inhibit the translation of target mRNA. , has a high degree of conservation, timing and tissue specificity, and currently plays an important role in the clinical diagnosis, treatment and prognosis evaluation of tumors, blood diseases, viral infec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12Q1/68
Inventor 李宁李启靖张永宏张卫红魏颖颖石佳李鹏
Owner 北京旷博生物技术股份有限公司
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