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

An extraction method and brain tissue technology, applied in the field of fish total RNA extraction, can solve the problems of difficult separation and purification of fish brain tissue, low total RNA content of fish brain tissue, and low RNA content, so as to eliminate nucleoprotein and Effects of interference from pigments, etc., shortening of processing time, and reduction of RNA degradation

Active Publication Date: 2014-12-17
BEIJING FISHERIES RES INST
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  • Summary
  • Abstract
  • Description
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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] The structure types of fish brain tissue are basically the same, and the content of different types of components is slightly different, but generally similar. The main components are lipids (phospholipids, glycolipids, cholesterol) and proteins. Lipids are not easy to remove during the process, and protein contamination is easy to form. The RNA content of fish brain tissue unit cells is relatively small, and RNA degradation is easily caused during sampling and operation, resulting in extraction failure.
Many problems make it difficult to isolate and purify the total RNA of fish brain tissue stably
[0004] At present, there are few methods for the targeted isolation and purification of total RNA from fish brain tissue. The total RNA extracted from fish brain tissue by traditional methods has low content, poor stability, and easy contamination, which cannot meet the requirements of subsequent experiments.

Method used

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

Examples

Experimental program
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Effect test

Embodiment 1

[0057] 1. Take 60 mg of fish brain 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).

[0058] 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. Centrifuge at 17,000 g for 10 min at 4°C for 8 s, and aspirate the supernatant into centrifuge tube A.

[0059] 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.

[0060] 3. Add 500μl 4mol / L ammonium sulfate (PH5.5) to centrifuge tube A, shake well, centrifuge at 5000g at 4°C for 3min, and suck the supernatant into centrifuge...

Embodiment 2

[0072] 1. Take 80 mg of fish brain 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).

[0073] 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. Centrifuge at 19,000 g for 10 min at 4°C for 10 s, and aspirate the supernatant into centrifuge tube A.

[0074] 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.

[0075] 3. Add 500μl 4mol / L ammonium sulfate (PH5.5) to centrifuge tube A, shake well, centrifuge at 7000g at 4°C for 5min, and aspirate the supernatant into cent...

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Abstract

The method provides a method for extracting total ribonucleic acid (RNA) of fish brain tissue. The method specifically includes subjecting the fish brain tissue to liquid nitrogen quick freezing, trizol lysate treatment, low-temperature high-speed centrifugation, and extraction by ammonium sulfate and chloroform n-butyl alcohol, adding isopropanol-sodium acetate for mixing, subjecting a mixture to adsorption and purification through an RNA purification and adsorption column, performing deoxyribonucleic acid (DNA) enzyme treatment, and performing washing to obtain total RNA of the fish brain tissue. According to the method for extracting the total RNA of the fish brain tissue, the influence of lipids (such as phospholipid, glycolipid, and cholesterol) and proteins in the fish brain tissue on RNA extraction is 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 brain 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] The structure types of fish brain tissue are basically the same, and there are slight differences in the content of different types of components, but they are generally similar. The main components are lipids (phospholipids, glycolipids, cholesterol) and proteins. Lipids ar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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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