DNAase step in differential display PCR

Richard R. Hardy rr_hardy at fccc.edu
Sat Jun 8 11:35:35 EST 1996


In article <lha-0806961046070001 at bullwinkle.paccm.pitt.edu>,
lha at med.pitt.edu (Louis Alarcon) wrote:

>In article <4pan98$n6 at neuro.usc.edu>, william at neuro.usc.edu (William Sun)
wrote:
>
>> After isolation of mRNA for differential display PCR, is it necessary to
>> treat the mRNA with DNAase?  I would like to omit extra steps if possible
>> to prevent RNAase contamination.
>
>
>If you are using anchored oligo-dTs as 3' primers for DD-PCR, it is not
>necessary to DNAse treat the starting RNA, since the oligo-dT will specify
>amplification of poly-A mRNA only.
>
>Lou Alarcon
>Dept of Surgery
>U of Pittsburgh

I really wish that this were so, but we saw MANY clones that were likely
from genomic DNA, where the 3' side had a short run (say 5-7) of A's.  We
ALWAYS treat with DNAase now.  Perhaps depends on the source of the RNA? 
Ours was from small numbers of primary lymphoid cells.  Make sure you read
the paper "Differental dismay", it covers a multitude of issues with DDRT.
____________________________________________________________________________
Richard R. Hardy                       Member, Institute for Cancer Research           
Fox Chase Cancer Center                Tel:    (215) 728-2463
7701 Burholme Ave.                     FAX:    (215) 728-2412
Philadelphia, PA 19111                 E-MAIL: RR_HARDY at fccc.edu



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