RNA extraction using trizol/tri

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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.

Contents

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

Steps

cell lysis

Image:Time required.png 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)

Image:Pause point.png 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

Image:Time required.png 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

Image:Optional step.png If supernatant appears turbid an additional chloroform cleaning step can be inserted here.

  • transfer aqueous upper phase into new tube

Image:Difficult_step.png 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

Image:Time required.png 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

Image:Time required.png 15-30 min depending on number of samples

  • wash pellet 70% EtOH (add & vortex briefly)
70% ethanol prepared with RNase-free water

Image:Optional step.png 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 Image:Critical_step.png 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 to resuspend pellet
  • 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 [1], chloroform is a narcotic [2])

See also

External links

Reagents

Protocols

Tips

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