A method for extracting RNA from serum exosomes

An extraction method and exosome technology, applied in the field of RNA extraction, can solve the problems of difficult extraction, high extraction cost, and small extraction amount, and achieve the effects of simple process, saving experimental cost and obvious effect.

Active Publication Date: 2018-03-02
GUANGZHOU SAGENE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] All in all, no matter which method is used, the isolation of exosomes in serum has always been difficult to extract, the amount of extraction is small, and the extraction cost is expensive.

Method used

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  • A method for extracting RNA from serum exosomes
  • A method for extracting RNA from serum exosomes
  • A method for extracting RNA from serum exosomes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Screening of Exosome-RNA extraction reagents

[0064] At present, the reagents used to extract exosomes on the market are mainly from two companies, System Biosciences and lifetechnology, so we compared the effects of the three products of the two companies on the exosome extraction of serum samples in this project, including ExoQuick from SBI (for blood) and ExoQuick-TC (for tissue and urine), Life Technology's Total Exosome Isolation Reagent (for serum).

[0065] The exosome extraction process of ExoQuick, ExoQuick-TC and Total Exosome Isolation Reagent is the same, the only difference is that the ratio of the amount of reagent used to the serum sample is different. The ratio of ExoQuick, ExoQuick-TC to serum samples is 60:250, while the ratio of Total Exosome Isolation Reagent is 50:250.

[0066] The method of exosome extraction using Total Exosome Isolation Reagent is:

[0067] (1) Place 250 μl of serum on ice to dissolve, then add 50 μl of Total Exosome Isolatio...

Embodiment 2

[0073] Optimization of Exosome-RNA extraction process

[0074] Due to the very high viscosity of the precipitated exosome, it is very difficult to deal with the RNA extraction and lysis step. The products of the above three manufacturers all need to be lysed for more than 30 minutes. Obviously, the water phase is less (only 100-150 μL), and the Ct values ​​of the final RNA detected by qPCR are all high. To this end, in order to optimize the exosome precipitate lysis step in the RNA extraction process, we used "life technology's Trizol LS 30min without ultrasonic treatment", "life technology's Trizol LS 5min with ultrasonic shock treatment" and "domestic Trigene reagent 5min add ultrasonic shock" these three different treatment methods, and compare the effect.

[0075] The specific operation of using Trigene reagent to extract exosome-RNA (that is, "domestic Trigene reagent added with ultrasonic vibration for 5 minutes") is as follows:

[0076] (1) Add 1ml Trigene reagent t...

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Abstract

The invention discloses an extracting method for RNA in serum exosomes. The extracting method is characterized by comprising the following steps that 1, the exosomes in serum are extracted; 2, the exosomes extracted in the step 1 are pyrolyzed through an RNA extracting reagent under the ultrasonic action; 3, the RNA of the exosomes pyrolyzed in the step 2 is separated into a water phase through chloroform; 4, the RNA obtained in the step 3 in the water phase is precipitated through isopropanol; 5, the RNA precipitated in the step 4 is washed through ethyl alcohol and then dissolved again, and the RNA in the serum exosomes is obtained. The extracting method for the RNA in the serum exosomes has the advantages of being short in extracting time, high in efficiency, good in effect and low in experimental cost.

Description

technical field [0001] The invention belongs to the field of RNA extraction, in particular to a method for extracting RNA from serum exosomes. Background technique [0002] Exosome (exosome) is a 30-100nm size, extremely low-density extracellular nanoscale capsule structure, which is formed by cells through a series of regulatory processes such as "endocytosis-fusion-efflux". Exosomes did not attract the attention of researchers for a long time after they were discovered. Until the introduction of nano-analysis technology and the announcement of the Nobel Prize in Biomedicine in 2013, this particle with a diameter of only tens of nanometers has received unprecedented attention. attention. [0003] According to research reports, the contents contained in exosomes such as microRNA (mRNA, miRNA), proteins and signaling complexes play an important role in the regulation of gene and protein expression in recipient cells. For example, miRNA (exosomal-miRNA) in serum exosomes has...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 曾宏彬罗宝花陈杰
Owner GUANGZHOU SAGENE BIOTECH
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