RNA extraction using trizol/tri
RNA extraction with TRIZOL (Invitrogen product name) or the equivalent TRI (Sigma-Aldrich product name) is a common method of total RNA extraction from cells. It takes slightly longer than column-based methods like RNAeasy but it has higher capacity and can yield more RNA.
- guanidinium isothiocyanate (powerful protein denaturant) -> inactivation of RNase
- acidic phenol/chloroform -> partitioning of RNA into aqueous supernatant for separation
Note: low pH is crucial since at neutral pH DNA not RNA partitions into the aqueous phase. Check the pH of old TRIZOL/TRI reagents!
- TRIzol Reagent (final concentration):
Phenol in saturated buffer (38 %) -- 380 ml/liter Guanidine thiocyanate (0.8 M) -- 118.16 g Ammonium thiocyanate (0.4 M) -- 76.12 g Sodium acetate, pH5 (0.1 M) -- 33.4 ml of 3M stock Glycerol -- 50 ml H2O to 1.0 liter
- 0.8 M sodium citrate / 1.2 M NaCl
- isopropanol (2-propanol)
- 75% EtOH in DEPC H2O
- RNase free water (filtered or DEPC)
draw water into RNase-free glass bottles add diethylpyrocarbonate (DEPC) to 0.01% (v/v) let stand overnight and autoclave
- RNA extraction (central, general page)
- RNA extraction with TRIZOL (Stanford)
- RNA extraction with TRIZOL (Uni Florida)
- Troubleshooting guide for TRIZOL extraction (Uni Toronto)