Extraction method of apostichopus japonicus body-wall total RNA

An extraction method, a technology of ginseng body wall, which is applied in the field of extraction of total RNA from the body wall of sea cucumber, can solve the problems of poor quality, low purity of total RNA, difficulty in meeting template purity, etc.

Inactive Publication Date: 2010-10-20
DALIAN OCEAN UNIV
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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 e...

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  • Extraction method of apostichopus japonicus body-wall total RNA
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  • Extraction method of apostichopus japonicus body-wall total RNA

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Embodiment Construction

[0024] a. Clean the temporarily raised imitation sea cucumber with seawater filtered by a 0.45μm filter membrane, and use a scalpel to take 50mg~100mg imitation sea cucumber body wall tissue and quickly put it into liquid nitrogen for quick freezing;

[0025] b. Grind the cryopreserved tissue into powder, and quickly put the powder into 1mL TRIzol lysis solution with a medicine spoon (the main component is phenol, but also 8-hydroxyquinoline, guanidine isothiocyanate, β-mercaptoethanol, etc. , Invitrogen company product) homogenize in a homogenizer, transfer the homogenate to centrifuge tube A, leave it at room temperature for 5 minutes, 4°C, 12000rpm / min for 10 minutes, and take the supernatant to centrifuge tube B;

[0026] c. Add 200μL of chloroform to centrifuge tube B, shake for 14-16s, let stand at room temperature for 2~3min, centrifuge at 4℃, 12000rpm / min for 15min, take the supernatant to centrifuge tube C;

[0027] d. Add 250μL of high-salt solution (a mixture of 0.8mol / L s...

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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...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 李霞秦艳杰王雪陈秋实郑柯
Owner DALIAN OCEAN UNIV
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