Hot/Acid Phenol Extraction
Michael G. Tencza
Michael G. Tencza
Sorry about the last bogus post, but I hit the wrong key somewhere. I would
like to see if anyone could clear up the following controversy. Recently
I became interested in RNA extractions and ran across a method using
phenol pH balanced to 5.2 with sodiom acetate and incubating the sample/phenol
mixture in a 55C water bath for 3-4 minutes prior to centrifugation. I
querried several sources about this procedure and obtained conflicting
information. One source told me that the acid pH of the phenol causes DNA
to end up in the organic phase and selectively leave the RNA in the
aqueous phase, while another said that both RNA and DNA end up in the
aqueous phase no matter the pH of the phenol). If anyone has experience
with this method I would appreciate a response to shed light on the
selectivity of this method. Thanking you in advance...
Mike
Bruce R. Troen
We make lots of RNA from mammalian cells in culture and use acid
phenol extraction with guanidinium isothiocyanate and then chloroform.
We prepare the phenol by equilibrating it with deionized water and
don't even both adjusting pH. I believe the extraction works because
the acidic pH permits a selective extraction of the RNA into the
aqueous phase (though this is not absolute). There are many protocols
that use this procedure (lots recently published in BioTechniques).
There are also several different commercial preparations of the GTC and
phenol available. We make our own.
If you need to be absolutely sure there is no DNA in the sample,
then you should treat with DNAase (for example, differential display -
PCR requires this). However we perform RT-PCR without any DNAase
treatment. Of course we also design our primers so that we can discern
if the amplified fragment contains extra material such as an intron.
Hope this helps.
Bruce R. Troen, M.D.
University of Michigan Medical School