Northern Blotting despair!
nishir at ohsu.edu
Thu Aug 21 19:35:46 EST 1997
We used to tear our hair out over northerns of low abundance RNA.
Being relative amateurs, we sat down and tried a bunch of differenct
protocols in parallel, systematically varying membrane types, transfer
methods, probing methods, etc. What we found was that the most
important variable was transfer method. The rapid (2 hr) downward
alkaline transfer (eg., "Turboblot") was far superior to conventional
upward capillary transfer (overnight) in SSC. Once we figured this
out, we went from using 10 ug of poly-A selected RNA to 10-20 ug of
total RNA per lane in order to see a signal. Other important factors
(though not as dramatic) were to use short (200-500 bp) riboprobes at
extremely high stringency. Obviously, high quality RNA helps, too.
The acidified guanidinium/ phenol method (eg., TriReagent) is great.
Rae Nishi, PhD
Dept. Cell & Developmental Biology
Oregon Health Sciences University
Portland Oregon 97201
**that's Orygun, NOT Ora-Gone**
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