RNA extraction using trizol/tri

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(Reagents)
(Principle)
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== Principle ==
== Principle ==
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 +
* '''guanidinium isothiocyanate''' (powerful protein denaturant) -> ''inactivation of RNase''
 +
* '''<u>acidic</u> 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 ==
== Reagents ==

Revision as of 14:21, 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 RNase
  • 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

Common mistakes

See also

External links

Reagents

Protocols

Tips

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