Reagents to be prepared: TRIzol (mainly phenol), chloroform (phenol: chloroform: Isoamyl alcohol=25:24:1), isopropanol, 80% alcohol (prepared with RNase free ddH2O), RNase free ddH2O.
Experimental process
1、 Sample Pretreatment
① 1 milliliter of whole blood and 1 milliliter of RNase free ddH2O.
② Centrifuge RNA blood preservation solution at 12000 rpm for 5 minutes, discard the supernatant, and add 1 mL of RNase free ddH2O.
③ 200 microliters of whole blood and 1 milliliter of RNase free dd H2O.
④ 500 microliters of whole blood and 1500 microliters of TRIzol (3 times the sample volume), thoroughly mix well.
All samples were placed at 15-30 ℃ for 5 minutes. ① ② ③ The sample was centrifuged at 12000 rpm for 10 minutes at 4 ℃.
2、 Homogenization
① ② ③ Discard the supernatant and add 1 milliliter of TRIzol, mix well. Place the samples ①, ②, ③, and ④ at room temperature for 5 minutes. Centrifuge at 12000 rpm for 10 minutes at 4 ℃. Take the supernatant into a new 2ml centrifuge tube, add 2 times the volume of phenol: chloroform: Isoamyl alcohol (25:24:1), shake it violently for 15s, place it at room temperature for 3min, and let it separate naturally.
Note: After adding TRIzol, if no further experiments are required, the sample can be stored at -70 ℃ for a long time.
3、 RNA precipitation
1. Centrifuge at 12000 rpm for 10 minutes at 4 ℃.
Note: The sample will be divided into three layers: the yellow organic phase, the middle layer, and the colorless aqueous phase. RNA is mainly present in the aqueous phase, transferring the aqueous phase to a new tube. Do not absorb the intermediate interface, otherwise it will cause DNA contamination in the RNA sample.
2. Suck the supernatant into a new centrifuge tube, add an equal volume of ice isopropanol, and let it sit at room temperature for 20-30 minutes. Centrifuge at 12000 rpm for 10 minutes at 4 ℃. RNA precipitates at the bottom of the tube.
4、 RNA Wash
1. Discard the liquid, add 80% alcohol of the same volume as TRIzol (prepared with RNase free ddH2O), slowly invert it up and down, and suspend and precipitate,
2. Centrifuge at 12000 rpm for 3 minutes at 4 ℃. Discard the liquid and let it sit at room temperature for 30 minutes (once the liquid has evaporated, it should not be left for too long as RNA samples are too dry and difficult to dissolve).
5、 Redissolving the RNA
Add 30-100 microliters of RNase free ddH2O to the sediment, blow with a gun and mix well- Long term storage at 70 ℃.