RNA extraction using trizol/tri

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== Steps ==
== Steps ==
 +
 +
 +
=== cell lysis ===
 +
 +
* (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)
 +
 +
=== phase separation ===
 +
 +
* 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
 +
 +
<pre>
 +
TRIZOL phases after chloroform addition
 +
 +
TOP - colourless aqueous phase (RNA) - 60% TRIZOL volume
 +
MIDDLE - interphase (DNA)
 +
BOTTOM - red (organic) phenol-chloroform phase w proteins & lipophilic molecules
 +
</pre>
 +
 +
=== RNA precipitation ===
 +
 +
* 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 ===
 +
 +
* 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
 +
* 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 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 ==
== Common mistakes ==

Revision as of 13:28, 14 February 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.

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

  • (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)

phase separation

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

RNA precipitation

  • 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

  • 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
  • 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 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

See also

External links

Reagents

Protocols

Tips

Personal tools