Northerns of large RNA?

[Patrick Hurban]

In article <4kh90c$2uu at post.its.mcw.edu>, kpleyte at post.its.mcw.edu (Kay A.
Pleyte) wrote:

>      I'm having problems getting a signal for a large RNA (possible 12 kb)
> on a Northern with a 32-P DNA probe.  My Northerns work OK for smaller
> RNAs.  It looks like the problem may be in the RNA extraction (breakage) or
> the transfer.  Any advice for working with large RNAs?
> 
> K.Pleyte
> 
> kpleyte at post.its.mcw.edu

The problem is most likely in the transfer.  I use a fairly standard
Formaldehyde gel protocol (MOPS buffer).  To aid in the transfer of large
RNAs, after running the gel I soak it in 50 mM NaOH, 100 mM NaCl for 45
min, followed by soaking in 100 mM Tris pH 7.5 for 30 min.  I then
transfer O/N in 20X SSC as usual.  This alkali treatment does not seem to
affect subsequent hybridization of probe but does apparently break the
larger RNAs into smaller chunks that transfer more readily.  I hope this
helps you out and good luck with your work.

-- 
Patrick Hurban