How to prepare CNS tissue for PCR?

nishir at ohsu.edu nishir at ohsu.edu
Thu Dec 9 18:26:47 EST 1993


In article <handley.1105794132A at 192.102.249.7> handley at ahelix.cerfnet.com (Dan
Handley) writes:
>I'm trying to find a good procedure for extracting RNA and DNA from brain
>tissue for PCR. Does anyone know if guanidinium/phenol-chloroform nucleic
>extraction (such as the Chomzsyski et. al. procedure for serum) will work on
>brain tissue? Or, can anyone suggest a good nucleic acid extraction
>procedure for brain tissue (because of the lipid content protease/detergent
>lysis buffer does not seem to work well on brain tissue). Many thanks for
>any suggestions!
>Alpha Helix Scientific Publications, Inc.
>11693 San Vicente Blvd. Ste 515
>Los Angeles, CA  90049
>(310) 854-5303
>
I have had good luck extracting DNA from rat brain tissue by using the
proteinase K/ chloroform phenol extraction procedure for genomic DNA in the
'red book' (Current Protocols in Molecular Biology).  You need to grind the
brain up well frozen in liquid nitrogen, digest overnight, and then repeatedly
extract with chloroform:phenol:isoamyl alcohol until the white stuff is gone
from the interface.  Then dialyze the DNA to remove the phenol.  We also
routinely extract RNA from rat and postmortem human brain with the acid
guanidinium extraction technique of Chomzsyski (we use the TriReagent that he
markets through MRC ) and we get much better yields than with other techniques.
 The lipid does not seem to be a problem..

Rae Nishi
OHSU
Portland OR



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