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MiRNA-122 detection kit and application thereof

A detection kit, miRNA-122 technology, applied in the biological field, can solve the problem of low specificity of liver function indicators, achieve the effect of reducing patient pain, excellent specificity, and reducing mistakes or errors

Inactive Publication Date: 2017-03-08
成都仕康美生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the specificity of these conventional liver function indicators is not high, and they are easily affected by other factors such as skeletal muscle injury, myositis, and strenuous exercise, etc., there is an urgent need for a more specific and sensitive detection marker for liver injury.

Method used

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  • MiRNA-122 detection kit and application thereof
  • MiRNA-122 detection kit and application thereof
  • MiRNA-122 detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: miRNA-122 detection kit

[0043] The detection kit is composed of 9ul reverse transcription buffer, 1ul reverse transcriptase, 17ul PCR buffer, 20ul ultrapure water, 200ul positive quality control, 200ul negative quality control and standard;

[0044] Wherein, the reverse transcription buffer is composed of 100mM dNTPs (with dTTP) 0.15ul, 10×RT Buffer1.5ul, 10mg / ml RNase inhibitor 0.2ul, 10uM reverse transcription primer 1ul and ultrapure water to form 6.15ul; The nucleotide sequence of the reverse transcription primer is shown in SEQ ID NO.1, specifically:

[0045] 5'-CGACTATCCTACGCAGGGTCCGAGGTATTCGCGTAGGATAGTCGCAAACACC-3';

[0046] Described reverse transcriptase is the MultiScribe that subpackages from ABI company TM Reverse Transcriptase, 50U / μL.

[0047] The PCR buffer is composed of 2×PCR Buffer 10ul (packed from the FastStartEssential DNA Green Master of Roche Company), 0.5ul of the upper primer for 10uM detection, 0.5ul of the lower primer for ...

Embodiment 2

[0055] Example 2: Detection of the expression level of miRNA-122 in plasma of patients after liver transplantation

[0056] 1. Sample collection and processing

[0057] 5 ml of blood from healthy people and liver transplant recipients were collected using ordinary EDTA blood collection tubes, 10 blood samples in total. Transfer the blood to a new 15ml centrifuge tube, centrifuge at 2000g for 10min at 4°C, and draw the upper layer of plasma into a new 15ml centrifuge tube. Then centrifuge the plasma at 4000g for 20min at 4°C, and draw the upper layer of plasma into a new 15ml centrifuge tube. Pipette 200ul and use QIAGEN's miRNeasy Serum / Plasma Kit to extract total RNA (including total microRNA) according to the operating instructions, and also extract equal volumes of positive and negative quality controls to obtain the final total RNA to be tested The volume is 40ul.

[0058] 2. Experimental detection

[0059] After the total RNA (including total microRNA) extracted from ...

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Abstract

The invention discloses a miRNA-122 detection kit, which comprises a reverse transcription primer, a detection primer, a quality control product and a standard product. The reverse transcription buffer solution and the PCR buffer solution provided by the kit are subjected to optimization on ingredients; the long-time stable storage can be favorably realized; meanwhile, the sampling workload can be simplified to the minimum, so that the fault or errors possibly occurring during the sample addition can be reduced; the stable and reliable detection result can be favorably obtained. The kit provided by the invention provides a novel liver injury detection method; the selected detection target miRNA-122 is only expressed on the liver, so that the specificity is superior to that of the prior art. The reagent kit provided by the invention is based on a fluorescent quantitative PCR (Polymerase Chain Reaction) platform; the non-invasive detection is realized; the pain of a patient is reduced; the detection sensitivity is high.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a miRNA-122 detection kit and its application. Background technique [0002] microRNAs are a class of non-coding single-stranded small molecule RNAs found in a variety of eukaryotic cells and viruses on endogenous chromosomes, with a length of 21-25 nt, which are highly conserved in evolution and can pass through their targets. Complementation of the 3' untranslated region (3'UTR) of the mRNA molecule leads to inhibition of translation of the mRNA molecule. miRNA is involved in regulating the expression of more than one-third of eukaryotic genes, inhibiting the translation of target genes, causing mRNA degradation, etc., and has very important biological functions. Further studies have shown that miRNAs play key roles in cell proliferation, differentiation and apoptosis, neuronal polarity, insulin secretion, brain morphogenesis, heart formation, embryonic development, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q2600/158C12Q2600/166C12Q2600/178
Inventor 魏亮卢俊龚雪
Owner 成都仕康美生物科技有限公司
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