2 replies to this topic

[SamCurt]

I'm extracting RNA for RNA-Seq, and I'm planning on using the TRIzol plus column cleanup method.

However, I noticed there are two ways this can be done:

• QIAgen recommends column-filtering ethanol/aqueous phase of Phenol/Chloroform Extraction, but
  
• Affymetrix recommends ethanol precipitate the RNA first, resuspend, add lysis buffer and ethanol, then column-filter.

So, which one is better? Thanks for the help!

[David C H]

I don't know that it matters.  Precipitating first will give you smaller, more manageable volumes.  To go from aqueous phase to column, I believe you need to add buffer RLT to the aqueous phase and then add EtOH so that the final volume may be in excess of 2ml (depending on the starting volume of your aqueous phase); you will need larger tubes and multiple spins to get it all through the column.  If you precipitate and resuspend in RLT, you can precipitate and resuspend in one tube with one spin through the column.

[SamCurt]

David C H, on Jul 7 2010, 08:16 AM, said:

To go from aqueous phase to column, I believe you need to add buffer RLT to the aqueous phase and then add EtOH so that the final volume may be in excess of 2ml

The aqueous phase still contain salts. So, according to QIAgen protocol, no RLT is needed-- just add equal volumes of EtOH. I use 100mg of tissues, though, and the aqueous phase that may come out of such an extraction will be 400uL, requiring me to load the column twice.