Resolution of small mRNA on Northerns

John S Jacobs Anderson jacobs at azstarnet.com
Thu Sep 4 20:02:19 EST 1997


(posted and emailed)
In article <1.5.4.32.19970904234407.002af30c at mailbox.uq.edu.au>,
B.Fletcher at MAILBOX.UQ.EDU.AU (Barb Fletcher) wrote:

>Hi!

Hi Barb..

>I am trying to get good resolution of a small (400-600bp) mRNA. I have tried
>some high resolution agarose (1.5%), and also 6% denaturing PAGE, but still
>get a broad band- (what I expect/ hope to get is several distinct bands of
>different sizes in that size range).
>I suspect I am seeing some degradation of my RNA, but all the usual tests-
>20S RNA/ 18S RNA, or probing with GAPDH, look fine. I guess they are more
>useful as measures of gross degradation- eg loss of 50bp will not be detected.
>
>So...., is there anyone else out there who also deals with smallish mRNA? Do
>you have similar problems? Could what I am seeing reflect a cellular
>process, eg variation in the length of poly A tail or shortening of the poly
>A tail prior to cellular degradation of the mRNA? 

I've done lots of work with mRNA in this size range (from cerevisiae). I
find that 6% PAGE run at 400 volts for 2-3 hours works fairly well. The
'fuzziness' that you see could very well be due to poly(A) -- if you're in
a metazoan system, tail lengths can vary quite a bit, so at steady state,
you get quite a bit of heterogenity.

Have you tried RNase H + oligo(dT) treatment to remove the tail? If it is
poly(A), that will take it right off and collapse the 'fuzz' down into a
tight band. If you need a RNase H treatment protocol, I can email you one.
If you try this, it's very important to use an 18 to 20-mer -- shorter
oligos don't seem to work as well.

Luck, 
John
--------------------------------------------------------------------
John S. J. Anderson                 http://www.azstarnet.com/~jacobs
Grad Student, MCB Dep¹t, U. of Az.       mailto:jacobs at azstarnet.com
What I tell you three times is true.



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