Trizol extraction for RNA and DNA

This is a protocol for harvesting both RNA and DNA from mammalian tissue. It is currently written for tissue preparation but can also be used on cell suspensions and cells grown on a monolayer.

Materials

• TRIzol or TRIzol LS reagent from Invitrogen
• 0.1M Sodium Citrate in 10% Ethanol (for 100ml 2.94g of the sodium citrate dihydrate mixed with 10ml EtOH and brought up to 100ml with H₂O)
• 100% Ethanol
• 75% Ethanol
• 8mM NaOH

RNA

Homoginize sample

• Homogonize tissue in 1ml trizol per 50-100 mg of tissue without having more than 10% sample volume compared to trizol
• Incubate sample 5' @ RT

Extract RNA

• Add 0.2ml of chloroform per 1mL of trizol and shake for 15"
• Spin at 12,000xg for 15' @ 4C
• Remove top aqueous phase (SAVE bottom organic phase for DNA)

RNA Precipitation

• Add 0.5ml of isopropanol per 1ml of trizol to the aqueous phase
• Incubate 10' @ RT
• Spin at 12,000xg for 10' @ 4C
• Wash pellet with 1ml 75% EtOH per 1ml trizol then vortex
• Spin at 12,000xg for 5' @ 4C

RNA Resuspension

• Air dry for 5-10' and resuspend in 30-50ul RNAse free H2O

DNA

Percipitation of DNA

• Take organic phase and carefully remove all aqueous phase
• Add 0.4ml 100% EtOH per 1ml of trizol mix by hand
• Incubate 2-3' @ RT
• Spin at 2,000xg for 5' @ 4C
• Remove sup (can be saved for protein purification)

DNA Wash

• Wash pellet 2x with 0.1M Sodium Citrate 10% EtOH 1ml per 1ml of trizol
• At each wash incubate in wash solution for 30' @ RT
• Spin at 2,000xg for 5' @ 4C
• Resuspend in 1.5-2.0ml 75% EtOH and incubate for 10-20' @ RT mix periodically
• Spin at 2,000xg for 5' @ 4C

Redissolve DNA

• Redissolve pellet in 300-600ul of 8mM NaOH per 50-70mg of tissue
• Spin at 12,000xg for 10 minutes to remove cell detritus
• Save supernatant with DNA