A kind of method for extracting total RNA from intermuscular bone of Dory
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An extraction method and intermuscular bone technology, applied in the direction of DNA preparation, recombinant DNA technology, etc., can solve the problems of inability to support molecular experiments, less total RNA in intermuscular bone, and extraction of total RNA has not yet occurred.
Inactive Publication Date: 2016-01-20
HUAZHONG AGRI UNIV
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The method of extracting total RNA from the intermuscular bone which is small in size, easy to degrade and easily contaminated by muscle protein has not yet appeared.
The amount of total RNA extracted from intermuscular bone by traditional methods is small and highly polluted, which cannot support further molecular experiments
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Embodiment 1
[0021] A method for extracting total RNA from the intermuscular bone of bream, the steps of which are:
[0022] 1) Sampling, using bream as the material: randomly select three bream sample fish of 200-300g, in order to prevent the blood from polluting the intermuscular bone, use a sterile syringe to draw 1.5-2.0ml of blood from the tail vein of bream 1-2 minutes after death, separate the intermuscular bone from the muscle of the fish and remove the muscle tissue, immediately place it in a sterilized pre-cooled mortar, and grind in the sterilized pre-cooled mortar successively while adding liquid nitrogen, Grind to powder. Take 50mg of intermuscular bone powder from each fish, put them into 2ml sterilized centrifuge tubes pre-filled with 1ml Trizol, shake well, and place at room temperature for 4 or 5 or 6 minutes;
[0023] 2) Centrifuge the placed centrifuge tube at 4°C and 12000rpm for 5min, transfer the supernatant to another 2ml sterilized centrifuge tube, add 1 / 5 volume o...
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Abstract
The invention discloses a method for extracting total RNA from the intermuscular bone of bream. The steps are as follows: 1) using bream as a material: take the intermuscular bone and place it in a sterilized and precooled mortar, grind it until the muscle Grind the interstitium into a powder and transfer it to a centrifuge tube, add Trizol to shake well, and place at room temperature; 2) Centrifuge at 4°C, take the supernatant, add 1 / 5 volume of chloroform, shake, and fully emulsify into a layer-free Uniform white emulsion, placed at room temperature; 3) Centrifuge at 4°C, take the supernatant and repeat extraction with chloroform; 4) Take the supernatant, add an equal volume of isopropanol, sodium citrate, and sodium chloride and mix well,- Stand still at 20°C; 5) After standing still, centrifuge at 4°C, discard the supernatant and leave the precipitate, add ethanol prepared with DEPC water, oscillate, and wash the precipitate repeatedly; 6) Centrifuge at 4°C, discard the supernatant and leave the precipitate, Let it dry naturally, add DEPC water to dissolve the RNA, and obtain a total RNA solution. The process is simple, the cost is low, and the total RNA obtained has high concentration, high purity, and good integrity, which meets the needs of further molecular biology research.
Description
technical field [0001] The invention relates to a method for extracting total RNA from fish bones, in particular to a method suitable for extracting total RNA from intermuscular bones of bream. This method is also applicable to the extraction of total RNA from the intermuscular bone of other fishes, and the extracted total RNA can be used for cDNA synthesis and gene expression research in further molecular biology. Background technique [0002] Fish meat is more and more popular because of its high nutritional content, low fat content, easy digestion, and is suitable for the general population. Dory ( Megalobrama amblycephala ) belongs to Cyprinidae ( Cypriniformes ), Cyprinidae ( Cyprinidae ), Subfamily Cichlidae ( Culterinae ), Bream ( Megalobrama ). It is one of the important herbivorous economic fishes unique to my country. Because of its wide feeding habits, low cost, fast growth, high survival rate, easy to catch, and has the advantages of delicious taste, sm...
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