How detect small size RNA in Northern analysis

Harold J Drabkin hdrabkin at mit.edu
Wed Nov 18 18:00:38 EST 1998


Jeongwon Park wrote:
> 
> Hi, Netters
> I want to know how to detect small size RNA in Northern analysis.
> My target RNA is 27 nt. I can't detect by general methods.
> Could you show me 'specific method' for detection of small size RNA in
> Northern analysis?
> 
I routinely probe for tRNA sized (75 nt) and half molecules.  You should
use Nytran Plus for the transfer; note the regular Nytran (small RNAs
won't stick to regular Nytran very well).  I transfer from 10-15% 7M
urea polyacrylamide gels, using standard Tris-Acetate buffer for two
hours, then bake the membrane at 80C for one hour.  
A prehyb is done for 2 hours in 10x denhardt's, 6x SSC, 1% SDS ar 42C,
and then I use a 5'-32P-labeled oligonucleotide as a probe (18-24 mer;
I'll give you a protcol for end-labeling to high SA if you need one;
about 3000 ci/mmole ), in 6X SSC, 1% SDS at 42C overnight.  Wash blot 2x
10 min in 6X SSC, the 2 X 10 min in 2x SSC.  No sheared DNA should be
used in the prehyb or hyb.  Background is very good.

Hope this helps

Harold J Drabkin



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