Protocol for Trizol RNA extraction
- Chloroform ( Fisher # C298-1)
- Sterile water RNAse-free ( Wisent 809-115-cl)
- 75% Ethanol
- Isopropanol ( Fisher # A419-4)
- Micro-centrifuge at 4?C
- Nanodrop spectrophotometer (Eppendorf)
- Trizol (Ambion # 15596026)
*Surfaces, pipettes and gloves should be wiped regularly with methanol to reduce RNAse contamination.
** Make sure that the tubes and tips you use were never touched without gloves.
*** Always work on ice (except when stated otherwise).
1. a) From a cell suspension:
- Centrifuge the cells at 3000 RPM for 5 minutes
- Aspirate the supernatant
- Add 1 ml of Trizol for 5-10 millions cells and resuspend the pellet as much as possible.
1.b) From adherent cells in a dish:
- Aspirate the media.
- Add 1 ml of Trizol on the dish and agitate to spread the Trizol uniformly.
- Transfer the lysed cells in a 1,5 ml tube.
2) Add 0,2 ml of chloroform per ml of Trizol used in the tubes and vortex them for 15 seconds.
3) Incubate the tubes 10-15 minutes at room temperature. Meanwhile, mix them a couple of times.
4) Centrifuge at 13 000 RPM at 4?C for 15 minutes.
5) Transfer the supernatant in another 1,5 ml tube.
6) Add 0,5 ml of isopropanol per milliliter of Trizol used.
7) Incubate for 10 minutes at -80?C.
8) Centrifuge at 13 000 RPM for 10 minutes at 4?C.
9) Discard the supernatant.
10) Wash the pellet with about 1 ml of 75% ethanol.
11) Centrifuge at 13 000 RPM at 4?C for 5 minutes.
12) Discard the supernatant.
13) Repeat steps 9 to 11.
14) Remove ethanol as much as possible and air dry the pellet (watch out not to dry it too much).
15) Smoothly resuspend the pellet in 16 ul of RNAse-free sterile water.
16) Heat the tubes at 60?C for 5 minutes to denaturate the RNA.
17) Put the tubes on ice.
18) Measure the RNA concentration and the 260/280 ratio using 1,5 ul of RNA on the Nanodrop. Attention: the Nanodrop must be on 10-15 minutes prior to use it.
19) Load about 1 ug of RNA on a 2% agarose gel to see the yield of the RNA.
20) Store the RNA at -80?C.