RNA extraction method

 At present, the Trizol method is commonly used to extract RNA from tissues or cells, and there are other methods such as: DNA extractor, RNA extractor, DNA extraction kit, RNA extraction kit and so on.

The Trizol method is a new type of total RNA extraction reagent, containing guanidine isothiocyanate and other substances, which can quickly disrupt cells and inhibit the nuclease released by the cells.

Although Trizol contains RNase inhibitors, 1ml of Trizol generally can only lyse up to 100mg of tissue. However, if the tissue is excessive, Trizol cannot completely infiltrate the tissue and cannot completely lyse the tissue. The RNase and other substances in the excess tissue will cause the degradation of RNA. This is a very important reason for RNA degradation.

At the same time, because RNA is easy to degrade, masks are generally required during the experiment to avoid tissue contamination. At the same time, the RNase-free imported pipette tips, EP tubes and DEPC water are selected. During the extraction process, you must always remind yourself of RNase Free-RNase Free-RNase Free, the experiment will be half successful~

Experimental extraction steps

The standard Trizol extraction steps are liquid nitrogen grinding-adding Trizol lysis-adding chloroform extraction-centrifugation-adding chloropropanol precipitation-centrifugation-alcohol washing-centrifugation.

1.Place the cell culture dish on ice, add Trizol to lyse for 5-10 minutes, pipette gently with a pipette tip, and suck the liquid into the imported EP tube.

2.Add 1/5 volume of chloroform, mix the liquid up and down, and let stand at 4°C for 10-15 minutes. (Chloroform is an organic solvent, which effectively separates the organic and inorganic phases quickly. The organic phase is mainly composed of phenol and protein binding, so that the protein and RNA are separated, and RNA enters the water phase).

3.Centrifuge at 4°C for 15 minutes. Be sure to choose low-temperature centrifugation. After centrifugation, it is divided into three layers. The RNA is in the supernatant, and the EP tube is gently taken out of the centrifuge to prevent the substance in the tube from shaking and causing the lower layer to agitate. When sucking the supernatant, be sure to act gently and avoid sucking too much. Generally, suck up to 400-500ul, avoid sucking the lower sediment, and put the liquid in a new EP tube.

4.Add an equal volume of isopropanol, let it stand at 4°C for 10 minutes, and then centrifuge at 12000 rpm for 10 minutes. Isopropanol is mainly used to precipitate RNA.

5.After removing the EP tube, you can see the sidewall sediment, gently aspirate and discard the supernatant.

6.Add 75% alcohol to the EP tube to wash the precipitate to help separate the remaining organic reagents (excessive organic reagents will affect the OD value and PCR reaction), flick the precipitate to float it in the alcohol, and let it stand for 1-2 minutes. Fully contact the precipitate, fully dissolve the organic reagents, and then centrifuge at 4°C for 5 minutes. Gently aspirate and discard the supernatant.

7.Put the EP tube in the centrifuge again for a short time centrifugation, and discard the remaining liquid on the tube wall. Then put the EP tube in a clean bench to dry for 5-10 minutes. Note that the RNA sample should not be too dry, otherwise it will be difficult to dissolve.

8.Add 50ul DEPC treated water, shake to fully dissolve the precipitate.

9.Measure RNA concentration. Store RNA at -80 degrees.

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