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Method for separating RNA (Ribonucleic Acid) from human serum/blood plasma sample and PCR (Polymerase Chain Reaction) verification method thereof

A serum and plasma technology, applied in the fields of biotechnology and medicine, can solve the problems of difficult RNA acquisition, low content, and low quality, and achieve the effects of improving extraction efficiency, accurate quantification, and simple and fast operation

Inactive Publication Date: 2013-04-24
ZHEJIANG SCI-TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

All these methods have encountered bottlenecks in the study of nucleic acids in blood due to the difficulty in obtaining RNA and its low content and low quality.

Method used

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  • Method for separating RNA (Ribonucleic Acid) from human serum/blood plasma sample and PCR (Polymerase Chain Reaction) verification method thereof
  • Method for separating RNA (Ribonucleic Acid) from human serum/blood plasma sample and PCR (Polymerase Chain Reaction) verification method thereof
  • Method for separating RNA (Ribonucleic Acid) from human serum/blood plasma sample and PCR (Polymerase Chain Reaction) verification method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1, a kind of method for isolating RNA from human serum, carries out following steps successively:

[0052] 1) Acquisition of serum samples:

[0053] ①. Pour 3 mL of peripheral blood (taken on an empty stomach in the morning) into a clean and dry centrifuge tube, immediately place it in a 37°C constant temperature incubator for 2 hours, and then place it in a 4°C refrigerator for 4 hours;

[0054] ②. Centrifuge the clot at 4°C, 4000rpm for 10min, and carefully pipette the supernatant into a 1.5mL centrifuge tube;

[0055] ③. The clinical serum samples separated in step 1) ② were mixed and stored in a -80°C refrigerator until use.

[0056] 2), heating pretreatment:

[0057] Select 250 μL of serum obtained in step 1) as a sample, heat at 75°C for 5 minutes, and then incubate at 42°C for 1 hour to obtain a pretreated serum sample;

[0058] 3) Add an equal volume of Trizol reagent to an RNase-free 1.5mL centrifuge tube to the above-mentioned pretreated serum sa...

Embodiment 2

[0062] Example 2, a method for isolating RNA from human serum, changing the pretreatment in step 2) to the following: protease pretreatment: 250 μL serum sample and 10 μg proteinase K were digested at 42° C. for 1 hour to obtain pretreated serum Sample; All the other are with embodiment 1.

Embodiment 3

[0063] Embodiment 3, a method for isolating RNA from human serum, change the pretreatment of step 2) to the following content: SDS pretreatment: 250 μ L serum sample is mixed with an equal volume of 10% SDS (mass concentration) solution, 4 ° C conditions Incubate for 1 h to obtain a pretreated serum sample; the rest are the same as in Example 1.

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Abstract

The invention discloses a method for separating RNA (Ribonucleic Acid) from a human serum / blood plasma sample, which sequentially comprises the steps of: 1, pretreating; 2, adding a Trizol reagent into the pretreated sample in an RNase-free centrifuge tube, uniformly mixing, standing at room temperature; 3, extracting chloroform; 4, adding isopropanol into the centrifuge pipe obtained from the step 3, uniformly mixing, standing at room temperature, centrifuging to obtain white sediment, washing and centrifuging the white sediment, and discarding supernatant; and 5, airing the sediment obtained from the step 4, adding diethyl pyrocarbonate for treating water to ensure that the sediment is dissolved to obtain a purified serum / blood serum RNA sample. The invention also discloses a method forcarrying out RT (Reverse Transcriptase)-fluorescence quantitative PCR (Polymerase Chain Reaction) verification by using the RNA obtained by the steps. The RNA with high quality and content can be effectively obtained by adopting the method of the invention.

Description

technical field [0001] The invention belongs to the fields of biotechnology and medicine, and relates to a method for quickly, economically and efficiently obtaining and verifying RNA from human serum samples, which provides convenience for the research of serum disease-related microRNA and has extensive scientific research value and clinical application prospects. Background technique [0002] Non-coding RNA (non-coding RNA, ncRNA) widely exists in organisms, does not encode protein, and can play an important role in many life processes in the form of RNA. With the revealing of more and more biological functions of ncRNAs, detection of changes in the expression profiles of ncRNAs in different physiological and pathological states has rapidly become a research hotspot. As a typical ncRNA, microRNAs (miRNAs) are a class of small RNA molecules with a length of 17-25nt. It is highly conserved, temporally and tissue-specific, and plays multiple roles in the regulation of cell g...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12Q1/68G01N21/64
Inventor 丁先锋郭江峰李艳徐立坚姚虎
Owner ZHEJIANG SCI-TECH UNIV
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