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Cirrhosis microRNA molecular marker composition and application thereof

A technology of molecular markers and uses, applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problem of no research on miRNA expression changes

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 in miRNA expression in patients with liver cirrhosis after medication have not yet been studied. In clinical and research, it is necessary to find miRNA markers that can effectively judge the efficacy of liver cirrhosis after medication and guide clinical drug withdrawal

Method used

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  • Cirrhosis microRNA molecular marker composition and application thereof
  • Cirrhosis microRNA molecular marker composition and application thereof
  • Cirrhosis microRNA molecular marker composition and application 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, 150 plasma samples from patients meeting the above definition of cirrhosis and 150 plasma samples from normal healthy subjects were pre-collected from Beijing You'an Hospital affiliated to Capital Medical University.

[0043] Peripheral venous blood was collected for 10 mi, anticoagulated with EDTA, and the plasma collection process was as follows: put the whole blood sample in a centrifuge at 4°C, and centrifuge at 1,500-3,000 g for 15 min. 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: 27) 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 determined using Thermo NanoDrop 2000c.

Embodiment 3

[0046] Example 3. Three-step method for quantitative detection of miRNA 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 system 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: 28) to each 20 μl system to monitor miRNA tailing and inversion 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'-CAGTGGTATCAACGCACTCCTTTTTT...

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Abstract

The invention discloses a cirrhosis microRNA molecular marker composition, with sequence No. ranging from 1 to 13, as well as application of the molecular marker composition in preparing a cirrhosis personalized medicine diagnostic reagent. Due to a significant difference between content of a microRNA molecular marker of a cirrhosis patient in plasma / serum and content in plasma / serum in health control, cirrhosis patient can be effectively distinguished from people of the health control; in a process of applying medicines to the cirrhosis patient, administration can be stopped by guiding and assistance, when cirrhosis outcome microRNA molecular marker is adjusted to a normal level. Furthermore, the invention discloses a diagnostic kit for guiding personalized administration of the cirrhosis. The liver cirrhosis outcome microRNA molecular marker disclosed by the invention, when being used for guiding personalized administration of cirrhosis patients, has characteristics of being simple to operate, safe and noninvasive, high in specificity, high in sensitivity and easy in massive screening.

Description

technical field [0001] The present invention relates to the field of individualized drug diagnosis of liver cirrhosis, in particular to a set of microRNA molecular marker combinations for liver cirrhosis, its use in diagnosis and / or prognosis assessment of patients with liver cirrhosis, and a kit containing its specific primers. It can be applied to the diagnosis and / or prognosis evaluation of patients with liver cirrhosis, and guides patients to scientifically administer and stop medication, which not only reduces the pain of medication for patients, but also reduces the waste of medical resources. 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 ...

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石佳李鹏魏颖颖
Owner 北京旷博生物技术股份有限公司
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