RNA extraction using trizol/tri-PDF

Overview

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 based on the research of Chomczynski P, Sacchi N. 1987 [1] and reviewed by the authors again in 2006 [2]. It takes slightly longer than column-based methods like RNAeasy b

ut it has higher capacity and can yield more RNA. Along with chaotropic lysis buffers it is generally considered the method that gives the best quality 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 or TRI reagent

If you want to make your own reagents, see here
RNA extraction using self-made guanidinium-acid-phenol reagents

• 0.8 M sodium citrate / 1.2 M NaCl
• isopropanol (2-propanol)
• chloroform
• 75% EtOH in DEPC H2O
• RNase free water (filtered or DEPC)

Steps

cell lysis

Cell lysis only takes a few minutes per well, but tissue homogenisation can take 10-20 minutes per sample depending on how tough the tissue is.

• (PBS wash)
• add trizol (cell lysis)

1ml / 3.5 cm diameter well (6-well)

5ml / 75 ml bottle

• homogenise by pipetting several times (mechanic lysis)

alternative for tubes: vortex 1 min

alternative for tissue: grind 1 g tissue in liquid nitrogen in a motar and pestle, put tissue into plastic screw-cap centrifuge tube + 15 ml TRIzol reagent, incubate samples for 5 min at room temp or 60° C (scaled up as needed)

• (5min at RT for complete dissociation of nucleoprotein complexes)

RNA is stable in trizol which deactivates RNases. You can take a break at this point keeping the sample in trizol for a short time or freezing it for a longer one.

phase separation

15-45 min depending on number of samples and whether an additional chloroform wash is necessary

• add chloroform (1/5 volume of trizol; e.g. 0.2ml to 1ml)
• shake for 15 sec (Eccles protocol: do not vortex)
• incubate 2-5 min at RT
• spin max. 12000g, 5-15 min, 2-8°C

if centrifugation hasn’t been sufficient the DNA-containing interphase will be cloud-like and poorly compacted

If supernatant appears turbid an additional chloroform cleaning step can be inserted here.

• transfer aqueous upper phase into new tube

Take care not to aspirate the DNA-containing white interface. This quickly happens and will lead to DNA contamination in your RNA prep.

TRIZOL phases after chloroform addition
 
TOP    - colourless aqueous phase              (RNA) - 60% TRIZOL volume
MIDDLE - interphase                            (DNA)
BOTTOM - red (organic) phenol-chloroform phase (proteins & lipids)

RNA precipitation and wash

20-40 min depending on number of samples

• add isopropanol (70% of aqueous phase or 1/2 trizol volume)
• 0.8 M sodium citrate or 1.2 M NaCl can be added

• (incubate 10min at RT)
• spin max g, 10-15 min, 4ºC
• remove supernatant
• (alternative RNA precipitation – RNeasy from Qiagen) better than alcohol precipitation for smaller amounts of RNA (less risk of losing a miniscule nucleic acid pellet); also reduces risk of organic solvent contamination

similar kits to RNeasy: MinElute kit, or Affymetrix sample clean-up

RNA wash

15-30 min depending on number of samples

• wash pellet 70% EtOH (add & vortex briefly)

70% ethanol prepared with RNase-free water

some prefer to wash the pellot more than once with 70% ethanol

• spin max g, 2-10 min, 4ºC
• air-dry pellet for 5-10 min Do not overdry the pellet or you won’t be able to redissolve it.

optional add RNase inhibitor

incubate at 55-60 C° for 10 min if hard to redissolve

• transfer to eppendorf tube
• spin 4° C, 5 min (to pellet undissolved material)

redissolving of RNA

• dissolve pellet in 50-100 µl filtered or DEPC H2O (note: DEPC inhibits RT reaction)
• alternatively, 0.5% SDS

pipetting up and down, heat to 55-60°C for 10 min

Common mistakes

• use too little trizol; very small volumes are hard to separate and will most likely lead to contamination
• aspirate some white interphase (DNA) when removing aqueous supernatant (RNA)
• use phenol/chloroform of the wrong pH (has to be acidic)
• not working under the hood (phenol is toxic , chloroform is a narcotic )