Can degraded RNA mimic mult. transcripts?

Loren Joseph ljlj at midway.uchicago.edu
Wed Jul 27 17:47:46 EST 1994


My colleague and I have identified a gene expressed in the cell line of
interest  which, in our first three northern blots, gave a gratifyingly
bright set of three bands in the range of 1.0 to 2.0 kb. We made the
mistake of doing a fourth blot, and saw only one VERY bright band. When we
looked back at the EtBr stained gels, the first three preparations clearly
had some RNA degradation as evidenced by a slight smearing of the ribosomal
bands and background, the 4th preparation was, of course, the best looking
RNA we've ever prepared.  There are also several low concentration but
distinct bands throughtout the 1-5 kb range of the first 3 preps.  We have
often seen these bands but ignored them since an actin probe indicated
intact mRNA.  

We are busily preparing a Northern with all our past preparations of RNA, 
hoping for a 'tiebreaker'. Perhaps the difference reflects the growth
conditions of the cells ( passage number/ time since feeding/etc). It would
be a disappointment not to have multiple transcripts, they showed
differential regulation.  Of course on the 4th blot we had different
experimental points in which expression was abolished except in the
control.  

Since I can't stand the suspense waiting for the next blot (probably blank
or overexposed), I ask the newsgroup.  Is there any evidence for sequence
or conformation specific cleavage of RNA on a limited scale during the
routine preparation of RNA samples? If this is possible, then probing with
actin might be an insufficient criterion for intact RNA. I have always
assumed that RNase degradation is not very discriminating.  Thinking back
to the multiple distinct low concentration bands we observed in RNA preps,
are those likely to just be mildly abundant intact transcripts or have I
been missing a clue about RNA degradation?  

Hopefully, you will tell me I am a dolt to even wonder about giving the
RNases so much credit.

																																	Thanks,

																																	Loren Joseph

ljlj at midway.uchicago.edu



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