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.

Principle

• guanidinium isothiocyanate (powerful protein denaturant) -> inactivation of RNases
• 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!

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)
• chloroform
• 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

See also

• RNA extraction (central, general page)

External links

Reagents

• TRIZOL reagent (Invitrogen), TRIZOL on wikipedia
• TRI reagent (Sigmal Aldrich)

Protocols

• RNA extraction with TRIZOL (Stanford)
• RNA extraction with TRIZOL (Uni Florida)
• Troubleshooting guide for TRIZOL extraction (Uni Toronto)

Tips

• Ambion tips on RNA extraction