The main components of Trizol reagent are guanidine isothiocyanate and phenol. Guanidine isothiocyanate can cleave cells, promote the dissociation of nucleosomes, separate RNA from protein, and release RNA into solution. When chloroform is added, it can extract acidic phenol, which can promote RNA to enter the aqueous phase. After centrifugation, it can form aqueous phase layer and organic layer, so that RNA can be separated from protein and DNA still remaining in the organic phase. The aqueous layer (colorless) is mainly RNA, and the organic layer (yellow) is mainly DNA and protein.
Experimental steps
After collecting biological materials, it is best to prepare RNA immediately. If temporary storage is required, biological materials shall be rapidly frozen with liquid ammonia and stored in -80 ° C freezer. When preparing RNA, take out the materials stored in the freezer and immediately break the cells by adding liquid nitrogen and grinding. Do not thaw first to avoid the role of RNase.
1. When extracting tissue RNA, the tissue was lysed with 1ml Trizol reagent every 50 ~ 100mg tissue; When extracting cellular RNA, first centrifuge and precipitate the cells, add 1ml Trizol to every 5-10 x106 cells, and then repeatedly blow with a gun or shake violently to lyse the cells;
2. Transfer the Trizol lysate of the above tissues or cells into the EP tube and place it at room temperature 15 ~ 30C for 5 minutes;
3. In the above EP tube, add chloroform according to the amount of 02ml chloroform per 1ml Trizol, cover the EP tube, shake vigorously in your hand for 15 seconds, place it at room temperature (15 ° C ~ 30 ° C) for 2 ~ 3 minutes, and then centrifuge 12000g (2 ° C ~ 8 ° C) for 15 minutes;
4. Take the upper aqueous phase and put it into a new EP tube. Add isopropanol according to the amount of 0.5ml isopropanol per 1ml Trizol, place it at room temperature (15 ° C ~ 30 ° C) for 10 minutes, and centrifuge 12000g (2 ° C ~ 8 * c) for 10 minutes;
5. Discard the supernatant, wash with 1ml 75% ethanol per 1ml Trizol, vortex mix, centrifuge 7500g (2 ° C ~ 8 * c) for 5 minutes, and discard the supernatant;
6. Allow the precipitated RNA to dry naturally at room temperature;
7. RNA precipitates were dissolved with RNase free water.
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