Extraction method of apostichopus japonicus body-wall total RNA

Abstract

The invention discloses a high-extraction-purity, good-integrity and high-yield extraction method of apostichopus japonicus body-wall total RNA (Ribonucleic Acid). The method comprises the following steps of: quickly freezing apostichopus japonicus body-wall tissue in liquid nitrogen; grinding and putting the frozen tissue in lysate to homogenate, centrifugate and take supernatant fluid; adding chloroform to the supernatant fluid and centrifugating to take the supernatant fluid to another centrifuge tube; then, adding a high-salt solution and isopropyl alcohol and filtering precipitation with ethanol; dissolving the precipitation with DEPC (diethypyrocarbonate) treated water to have constant volume; sequentially adding a DNA enzyme buffer solution without RNA enzymes, DNA enzymes without RNA enzymes and an RNA enzyme inhibitor to the dissolved solution to mix; carrying out a water bath at 37 DEG C to obtain DNA lysate; adding phenol and chloroform in a ratio of 5:1 to the DNA lysate to mix, centrifugate and take supernatant fluid; adding a glycogen solution, a potassium acetate solution and pre-cooling ethanol to the supernatant fluid; mixing and staying over night; centrifugating to discard the supernatant fluid; washing and dying the precipitation with ethanol; dissolving the solution with the DEPC treated water to have constant volume of 20 mu l; and storing at 80 DEG C below zero.

Description

Technical field: [0001] The invention relates to a method for extracting total RNA from the body wall of a sea cucumber, in particular to a method that can avoid the influence of mucopolysaccharides, collagen and pigments in the body wall of the sea cucumber with high extraction purity, good integrity and high yield A method to imitate the total RNA of the sea cucumber body wall. Background technique: [0002] Extracting RNA with high purity and integrity from tissue cells is a necessary prerequisite and key for molecular biology research. Because of the differences in tissue composition and structure of different species, the extraction of RNA from different cell sources must be selected accordingly. Preparation. [0003] The connective tissue layer of the body wall of the sea cucumber is thicker and consists of loosely distributed collagen fibers. The chemical components of the body wall are mainly mucopolysaccharide, collagen and pigment. This makes it difficult to homogenize t...

AI Technical Summary

Problems solved by technology

[0003] The connective tissue layer of the body wall of A. japonicus is relatively thick, which is composed of loosely distributed collagen fibers. The chemical components of the body wall are mainly mucopolysaccharides, collagen and pigments, which makes it difficult to homogenize samples when extracting total RNA from the body wall of A. japonicus. , the extracts obtained by conventional RNA extraction methods often contain colloidal insolubles such as mucopolysaccharides and a large amount of dark brown pigments. The purity of total RNA is low and the quality is poor. These impurities not only seriously affect subsequent enzymatic reactions such as RT-PCR , and it is also difficult to meet the requirements for template purity in some experiments (such as DDRT-PCR, molecular cloning, etc.)

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