Method for extracting micro ribonucleic acid from serum

A microRNA and serum technology, applied in the field of microRNA extraction, can solve the problems of serum miRNA loss, miRNA loss, high cost of separation column method, and achieve the effects of low extraction cost, improved detection level, and good application prospect.

Inactive Publication Date: 2011-10-26
FUWAI HOSPITAL OF CARDIOVASCULAR DESEASE CHINESE ACAD OF MEDICAL SCI
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Problems solved by technology

For example, miRNA is much smaller than mRNA in length, and miRNA mainly exists in the form of protein complexes in cells; conventional TRIzol extraction methods will cause a large loss of serum miRNA
The separation column extraction method for miRNA can ...

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  • Method for extracting micro ribonucleic acid from serum

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Embodiment Construction

[0020] Take 500 μl plasma or serum and add 500 μl TRIzol (Invitrogen, the main components of the reagent are phenol, 8-hydroxyquinoline, guanidine isothiocyanate, β-mercaptoethanol, etc.), vortex shaker vigorously for 30 seconds, and stand at room temperature for 5 minutes; Add 100 μl of isopropanol, mix by inverting, vortex shaker vigorously for 30s until the liquid is transparent, let stand at room temperature for 5min; centrifuge at 3000rpm at 4℃ for 10min, transfer the supernatant to a new centrifuge tube; Add 500 μl chloroform to the supernatant, mix it upside down, centrifuge at 13,000 rpm at 4°C for 10 min, then transfer the supernatant to a new centrifuge tube; Mix upside down and let stand at room temperature for 10 minutes; centrifuge at 13,000 rpm at 4°C for 10 minutes to precipitate RNA; absorb ethanol and dry RNA; dissolve RNA in 30 μl DEPC (diethyl pyrocarbonate) water, and bathe in 58°C for 10 minutes to obtain the extract of the present invention.

[0021] In a...

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Abstract

The invention provides a method for extracting micro ribonucleic acid (miRNA) from serum. The method comprises the steps that: 500[mu]l of TRIzol is added to 500[mu]l of blood plasma or serum, the mixture is violently concussed for 30s by using a vortex oscillator, and the mixture is then stood under room temperature for 5min; 100[mu]l of isopropanol is added to the mixture, the mixture is well-mixed by tumbling, then the mixture is violently concussed for 30s by using a vortex oscillator until the liquid turns transparent, and the liquid is stood under room temperature for 5min; the liquid is separated for 10min by using a centrifuge under a temperature of 4 DEG C with a speed of 3000rpm, and the supernatant is transferred into a new centrifuge tube; 500[mu]l of chloroform is added to the supernatant in the centrifuge tube, and the liquid is well-mixed by tumbling, then the liquid is separated for 10min by using a centrifuge under a temperature of 4 DEG C with a speed of 13000rpm, and the supernatant is transferred into a new centrifuge tube; ethanol with a volume which is 2.5 times than the supernatant in the centrifuge tube is added to the centrifuge tube, the liquid is well-mixed by tumbling, and the liquid is then stood under room temperature for 10min; the liquid is separated for 10min by using a centrifuge under a temperature of 4 DEG C with a speed of 13000rpm, and then RNA is precipitated; ethanol is removed by absorption, and RNA is dried; the obtained RNA is dissolved with 30[mu]l of DEPC-treated water, and the solution is processed through water-bath for 10min at a temperature of 58 DEG C, such that a target extract is obtained. The method provided by the present invention is simple and applicable. With the method, serum miRNA loss during extraction can be avoided. The method also has advantages of low extraction cost, fast speed and high extraction rate.

Description

technical field [0001] The invention belongs to a method for extracting microRNA from serum. Background technique [0002] miRNA (microRNA micro ribonucleic acid) is a type of endogenous single-stranded non-coding RNA (ribonucleic acid), generally composed of 21 nucleotides. The Sanger miRNA database version 16.0 released on September 10, 2010 contains 17,341 mature miRNA sequences from 142 species, including 1,048 miRNAs that have been cloned from human cells (http: / / www.mirbase.org) . As micro-regulatory molecules, miRNA specifically binds to the 3'-UTR of mRNA (messenger ribonucleic acid), inhibits protein translation or directly causes mRNA degradation, and regulates protein expression at the post-transcriptional level. Bioinformatics prediction results show that the translation activity of about 50% of protein-coding genes in mammals is regulated by miRNA. Functional studies have shown that miRNAs are involved in the regulation of almost all cell life processes, and ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 聂宇陈曦丛祥凤侯剑峰张洋顾海勇
Owner FUWAI HOSPITAL OF CARDIOVASCULAR DESEASE CHINESE ACAD OF MEDICAL SCI
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