Transportation temperature: normal temperature transportation

Operation steps: 1 Sample processing: a. Plant tissue: Take fresh or -70 ℃ frozen 100mg tissue and grind it in liquid nitrogen. Add the powder to 1ml lysis buffer and mix well.

b. Animal tissue: Take fresh or -70 ℃ frozen 100mg tissue and add 1ml lysis buffer. Use a tissue grinding pestle or homogenizer to homogenize the tissue.

c. Adhering cells: directly add lysis buffer to the culture plate to lyse cells, adding 1ml lysis buffer for every 106 cells. Use a sampler to blow and mix well.

d. Cell suspension: Collect cells by centrifugation. Mix 1ml lysis buffer for every 106 animal, plant, and yeast cells or every 107 bacterial cells.

e. Blood processing: Take 0.2-1ml of fresh blood and add 3 times the volume of red blood cell lysate. Mix well and let it stand at room temperature for 10 minutes. Centrifuge at 10000rpm for 1 minute. Discard the supernatant. If the precipitate contains red blood cells, add twice the volume of red blood cell lysis buffer and repeat the lysis steps. After centrifugation, add 1 ml of lysis buffer to the precipitate and mix well.

2. Leave the processed sample at room temperature for 5 minutes to completely separate the nucleic acid protein complex.

3. Add 0.2ml chloroform to the homogenized sample, cover the tube, vigorously shake for 15 seconds, and let it stand at room temperature for 3-5 minutes.

Centrifuge at 12000 rpm for 10 minutes at 2-8 ℃. RNA mainly exists in the colorless aqueous phase in the upper layer. Transfer the aqueous phase to a new tube and do not absorb the precipitate.

5. Pre treatment of adsorption column: Add 500ul of column washing solution to the adsorption column, let it stand at room temperature for 2 minutes, centrifuge at 12000 rpm at 2-8 ℃ for 2 minutes, and discard the waste liquid.

6. Add 200ul anhydrous ethanol to the supernatant collected in step 4, mix well, add it to the adsorption column and let it stand for 2 minutes. Centrifuge at 12000rpm at 2-8 ℃ for 2 minutes, and discard the waste liquid.

7. Add 600ul of rinsing solution to the adsorption column (please check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm at 2-8 ℃ for 2 minutes, and discard the waste liquid.

8. Add 600ul of rinse solution to the adsorption column, centrifuge at 12000 rpm at 2-8 ℃ for 2 minutes, and discard the waste liquid.

Centrifuge at 12000rpm for 2 minutes, discard the collection tube, and place the adsorption column at room temperature for a few minutes to remove any residual rinse solution from the column.

10. Place the adsorption column into a new tube and add 50-100ul of RNase free ddH2O dropwise to the center of the membrane. Leave it at room temperature for 5 minutes and centrifuge at 12000rpm for 2 minutes to obtain RNA.

matters needing attention:

1. All related utensils and consumables should be RNase free products, and the operation process should be careful. Wear masks and gloves to avoid sample contamination by RNase in the environment.

2. The OD value of RNA in aqueous solution may be between 1.5-1.9, but this does not mean that RNA is impure and needs to be detected by electrophoresis.