Method for extracting RNA from extracellular vesicles

Abstract

The invention relates to a method for extracting RNA from extracellular vesicles. The method comprises the following steps: S1, separating and purifying the extracellular vesicles in a biological sample, and cracking the separated and purified extracellular vesicles; S2, adding an organic solvent to enable RNA to enter a water phase; S3, adding an alcohol organic solvent to the RNA containing water phase obtained in step S2; S4, adding a cation containing reagent for binding with negatively charged RNA; S5, adding a solution containing an inert coprecipitation reagent component; S6, centrifuging the solution obtained in step S5 to obtain RNA precipitate; S7: washing the precipitate and redissolving the precipitate to obtain an extracellular vesicle RNA solution. The method for extracting RNA has the advantages of short extraction time, low cost, high yield, high purity, good effect and the like, and facilitates subsequent tests.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for extracting RNA in extracellular vesicles. Background technique [0002] Extracellular vesicles refer to all membrane vesicle structures wrapped by phospholipid bilayer structure released by cells into the external environment. Common extracellular vesicles include exosomes, microvesicles, apoptotic bodies, and bacterial outer membrane vesicles. [0003] The principle of the current conventional extracellular vesicle RNA extraction method is to use phenol as the main extraction reagent supplemented with guanidine isothiocyanate or β-mercaptoethanol to depolymerize proteins and nucleic acids and denature proteins at the same time, because phenol is not enough to inhibit RNA Enzymatic activity therefore requires the use of guanidine isothiocyanate or β-mercaptoethanol to denature RNases. Then add chloroform and other reagents to extract the RNA in the sample and then cent...

AI Technical Summary

Problems solved by technology

[0004] However, since extracellular vesicle samples are not easy to obtain, and generally the amount is very small, the above-mentioned traditional method for extracting extracellular vesicle RNA has the following disadvantages: 1. In step 4, isopropanol captures the surrounding RNA Combine water to promote RNA aggregation. Since the RNA content in extracellular vesicle samples is small, and RNA is slightly soluble in water, isopropanol alone is not enough to precipitate the RNA in the system. This step will bring great Proportional RNA loss, or even RNA extraction failure

2. In step 6, due to the small amount of RNA precipitation without the protection of other substances, and because the RNA is slightly soluble in water, the trace amount of RNA is directly soluble in water during the washing process, resulting in RNA extraction failure

3. In step 6 and step 7, the RNA is precipitated by centrifugation, but since the sample of extracellular vesicles is usually very small, the amount of RNA extracted is also very small, and the precipitation is usually not visible. The RNA precipitate was sucked away and discarded, which caused RNA extraction failure

Images