Method for extracting micro-RNA from plasma and kit thereof

A technology of plasma and reagents, which is applied in the field of methods and kits to extract microRNA, can solve the problems of high price and low extraction volume, and achieve the effect of improving recovery efficiency

Inactive Publication Date: 2017-01-11
深圳市人口和计划生育科学研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the extraction method of plasma miRNA mainly uses imported kits from abroad, but the kits for extracting plasma miRNA have the disadvantages of high price and low extraction volume, and the total amount of extracted RNA often cannot meet the requirements of subsequent experiments

Method used

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  • Method for extracting micro-RNA from plasma and kit thereof
  • Method for extracting micro-RNA from plasma and kit thereof
  • Method for extracting micro-RNA from plasma and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] A method for extracting microRNA in human plasma, according to the following steps:

[0043] (1) After collecting human anticoagulated whole blood and mixing it, centrifuge at 500rpm for 10 minutes to remove blood cells, and obtain plasma samples and store them at -80°C for later use.

[0044] (2) Take 200 μL of the above plasma sample in a 1.5 mL centrifuge tube, add 200 μL of 10% sodium dodecyl sulfate (SDS) for 1:1 mixing, then add 1 μL of proteinase K 20 mg / ml, and heat at 75°C for 5 minutes; ℃ warm bath digestion 1h.

[0045] (3) Add 2 times the volume of Trizol reagent to the treated sample, mix well, and let stand at room temperature for 5 minutes; add 20% volume of chloroform, shake for 15 seconds, let stand for 2 minutes; centrifuge at 12,000 rpm and 4°C for 15 minutes, and take the supernatant .

[0046] (4) Add an equal volume of isopropanol and 1 μL of 15 mg / ml glycogen, mix well, and let stand overnight at -80°C.

[0047] (5) Centrifuge at 12000rpm at 4°...

Embodiment 2

[0050] A method for extracting microRNA in human plasma, according to the following steps:

[0051] (1) After collecting human anticoagulated whole blood and mixing it, centrifuge at 500rpm for 10 minutes to remove blood cells, and obtain plasma samples and store them at -80°C for later use.

[0052] (2) Take 200 μL of the above plasma sample into a 1.5 mL centrifuge tube, add 200 μL of 10% sodium dodecyl sulfate (SDS), and add 1 μL of proteinase K 20 mg / ml at the same time, shake and mix, heat at 75 °C for 5 minutes, and then heat at 42 °C Incubate for 1 hour.

[0053] (3) Add 2 times the volume of Trizol reagent to the treated sample, mix well, and let stand at room temperature for 5 minutes; add 20% volume of chloroform, shake for 15 seconds, let stand for 2 minutes; centrifuge at 12,000 rpm and 4°C for 15 minutes, and take the supernatant .

[0054] (4) Add an equal volume of isopropanol and 1 μL of 15 mg / ml glycogen, mix well, and let stand at -80°C overnight (8-12 hour...

Embodiment 3

[0058] A method for extracting microRNA in human plasma, according to the following steps:

[0059] (1) Collect 200 μL of human plasma, add 1 μL of proteinase K 20 mg / ml and an equal volume of 10% SDS, shake and mix.

[0060] (2) Heating at 75°C for 5 minutes, then incubating at 42°C for 1 hour.

[0061] (3) Add 2 times the volume of Trisol reagent and 1 / 5 times the volume of chloroform, shake and mix, and let stand for 15 minutes.

[0062] (4) Centrifuge at 12,000 rpm for 15 minutes at 4°C, take the supernatant, add an equal volume of isopropanol and 1 μL of 15 mg / ml glycogen.

[0063] (5) Stand overnight at -80°C, centrifuge at 12,000 rpm for 10 minutes at 4°C.

[0064] (6) The precipitate was collected, washed with 75% ethanol, and centrifuged at 12000 rpm for 5 minutes at 4°C.

[0065] (7) Collect the precipitate, dry it at room temperature, add 14 μL RNase-free water to dissolve it, and store it at -80°C.

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Abstract

The invention relates to the technical field of biology, in particular to a method for extracting mciro-RNA from plasma and a kit thereof. The method comprises the steps that a lysate is added into a plasma sample, and a protein removal solution is added; an RNA extraction reagent is added for performing RNA extraction, then, chloroform is added for oscillation mixing, and centrifuging is performed to collect supernatant liquid; a precipitating agent and a settling agent are added, and centrifuging is performed to collect sediments; a washing solution is used for performing washing, centrifuging is performed to collect sediments, RNase-free water is used for performing dissolution. According to the extraction method, firstly, sample cracking and protein removal are performed, and then RNA extraction is performed, so that proteins combined with the micro-RNA are modified to release the micro-RNA, and the integrity of the micro-RNA is kept. According to the kit, the lysate and the protein removal solution are used at the same time for helping sufficient degradation of proteins combined with the micro-RNA, and the precipitant isopropanol and the settling agent glycogen are used at the same time, so that the micro-RNA recycling efficiency is greatly improved.

Description

technical field [0001] The application relates to the field of biotechnology, in particular to a method for extracting microRNA from blood plasma and a kit thereof. Background technique [0002] In recent years, with the continuous development of emerging molecular biology research technologies such as qPCR and RNA-seq, more in-depth research on life events at the RNA level has gradually become the focus of life sciences. MicroRNAs (MiRNAs, miRNAs) are a class of small molecule non-coding RNAs with a length of 17-25 bases, which negatively regulate the expression of target genes through complementary binding to target gene transcripts, or called gene silencing. . This gene silencing effect plays an important role in various physiological and pathological processes such as signal transduction, stem cell development, tumor development, infection and immunity. Studies have shown that miRNA genes account for 3%-5% of the total number of predicted genes, and regulate about 20%-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003C12Q2523/32
Inventor 洪文旭胡娜段山林圣曾序春邵豪
Owner 深圳市人口和计划生育科学研究所
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