Method for extracting total ribonucleic acid (RNA) of fish testicle tissue

An extraction method, testis technology, applied in the field of fish total RNA extraction, can solve the problems of insufficient grinding of fish testis tissue, low concentration of total RNA, protein pollution, etc., to eliminate interference from nucleoproteins and pigments, etc., and inhibit internal Source RNase activity, effect of inhibiting endogenous RNase

Inactive Publication Date: 2014-12-17
BEIJING FISHERIES RES INST
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0002] Total RNA separation and purification technology is the basis of molecular biology research technology. The separation and purification method of extracting total RNA with stable purity, good integrity, high repeatability and no pollution from tissues is the key to determining the quality of subsequent experimental results. Some specific The general RNA separation and purification method used for the site often cannot meet the requirements, and corresponding processing and extraction preparation methods are required
[0003] Fish testis tissue contains a large amount of protein components (spermatogonia, spermatocytes, sperm, etc.) and connective tissue. Conventional RNA separation and purification methods are easy to protein contamination during extraction, and samples are not easy to grind during operation. , it is easy to produce viscous micelles during lysis, resulting in the degradation and low yield of RNA during the extraction process
Due to the above problems, it is difficult to isolate and purify the total RNA of fish testis tissue stably
[0004] At present, there are few targeted methods for isolating and purifying total RNA from fish testis tissue. Traditional methods are used to treat fish testis tissue with insufficient grinding, cracking and easy formation of viscous micelles, low concentration of extracted total RNA, poor stability, and high contamination rate. , unable to meet the requirements of subsequent experiments

Method used

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  • Method for extracting total ribonucleic acid (RNA) of fish testicle tissue
  • Method for extracting total ribonucleic acid (RNA) of fish testicle tissue

Examples

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Embodiment 1

[0052] The specific steps of the method for extracting total RNA from fish testis tissue described in this embodiment are as follows:

[0053] 1. Take 60 mg of fish testis tissue with surgical tools, wash it with sterile RNase-free (RNase-free) water, quickly put it into liquid nitrogen for quick freezing, and store it at ultra-low temperature (-80°C).

[0054] 2. Quickly transfer the frozen sample into an RNase-free EP tube containing 1ml Trizol lysate, use an RNase-free electric homogenizer to homogenize, place the EP tube in ice, and ultrasonically break for 8 seconds with an interval of 15 seconds. 8s, filter once with a 35-mesh filter, centrifuge at 170004°C for 5min, and suck the supernatant into centrifuge tube A.

[0055] Note: Trizol lysate (the main component of TRIzol is phenol, also contains guanidine isothiocyanate, sodium lauryl sarcosinate, sodium acetate, etc., Life technologies company), add 25 μl β-mercaptoethanol and 0.5 μl of 8-hydroxyquinoline.

[0056] ...

Embodiment 2

[0068] The specific steps of the method for extracting total RNA from fish testis tissue described in this embodiment are as follows:

[0069] 1. Take 80 mg of fish testis tissue with surgical tools, wash it with sterile RNase-free (RNase-free) water, quickly put it into liquid nitrogen for quick freezing, and store it at ultra-low temperature (-80°C).

[0070] 2. Quickly transfer the frozen sample into an RNase-free EP tube containing 1ml Trizol lysate, use an RNase-free electric homogenizer to homogenize, place the EP tube in ice, and ultrasonically break for 10 seconds with an interval of 20 seconds. 10s, filter once with a 40-mesh filter, centrifuge at 19,000g at 4°C for 5min, and aspirate the supernatant into centrifuge tube A.

[0071] Note: Trizol lysate (the main component of TRIzol is phenol, also contains guanidine isothiocyanate, sodium lauryl sarcosinate, sodium acetate, etc., Life technologies company), add 25 μl β-mercaptoethanol and 0.5 μl of 8-hydroxyquinoline...

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Abstract

The invention provides a method for extracting total ribonucleic acid (RNA) of fish testicle tissue. The method includes subjecting the fish testicle tissue to liquid nitrogen quick freezing, trizol lysate treatment, uniform stirring, filtering, centrifugation, digestion by protease K, and extraction by chloroform n-butyl alcohol, precipitation by isopropanol-sodium acetate, performing deoxyribonucleic acid (DNA) enzyme treatment, and performing washing to obtain the total RNA of the fish brain tissue. According to the method for extracting the total RNA of the fish testicle tissue, the influence of a large amount of protein components and connective tissue in the fish testicle tissue can be avoided, and the RNA separation and purification method is stable in purity, good in integrity, high in repeatability, and free of pollution.

Description

technical field [0001] The invention relates to the extraction of fish total RNA, in particular to a method for extracting fish testis tissue total RNA. Background technique [0002] Total RNA separation and purification technology is the basis of molecular biology research technology. The separation and purification method of extracting total RNA with stable purity, good integrity, high repeatability and no pollution from tissues is the key to determining the quality of subsequent experimental results. Some specific The general RNA isolation and purification method used for the site often cannot meet the requirements, and corresponding processing and extraction preparation methods are required. [0003] Fish testis tissue contains a large amount of protein components (spermatogonia, spermatocytes, sperm, etc.) and connective tissue. Conventional RNA separation and purification methods are easy to protein contamination during extraction, and samples are not easy to grind dur...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 杨晓飞徐绍刚李文通袁丁杨贵强马峻峰
Owner BEIJING FISHERIES RES INST
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