I've been successfully doing Northerns for some time looking at mRNAs
which range in size from 1-12kb. The largest mRNA (12kb) has the lowest
expression level. Recently I lost the ability to detect this message.
Using newly prepared probe (both cDNA and riboprobe) did not change the
situation. I now suspect that the transfer procedure from gel to
membrane is to blame. However I have not altered my protocol, which is
to do a capillary blot O/N from 10XSSC to a positively charged nylon
membrane (Boehringer Membrane). All other targets I look for are
present in expected quantities. Has anyone else experienced problems
with transfer of higher molecular weight RNA? If so what are your
suggestions?
Science is such fun!!
Helen
