RNA extraction using trizol/tri: Difference between revisions
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=== cell lysis === | === 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) | * (PBS wash) | ||
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=== phase separation === | === 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) | * add chloroform (1/5 volume of trizol; e.g. 0.2ml to 1ml) | ||
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</pre> | </pre> | ||
=== RNA precipitation === | === 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) | * add isopropanol (70% of aqueous phase or 1/2 trizol volume) | ||
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=== RNA wash === | === RNA wash === | ||
[[Image:Time required.png]] 15-30 min depending on number of samples | |||
* wash pellet 70% EtOH (add & vortex briefly) | * wash pellet 70% EtOH (add & vortex briefly) |
Revision as of 05:03, 6 May 2008
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
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
- 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
- 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 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
- 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