Better methods for PolyA+ please

frist at ccu.umanitoba.ca frist at ccu.umanitoba.ca
Tue May 28 10:04:39 EST 1991


In article <1991May28.165855.1 at rsbs0.anu.edu.au> jjw at rsbs0.anu.edu.au writes:
>I'm posting this for a friend in Melbourne, Australia who doesn't have 
>easy net access.
>
>********************************************************************************
>Advice needed on easy methods for Poly A+ RNA isolations (from soft mammalian
>tissue).
{ ... details of request deleted}
>
>Jeremy Weinman		Plant Microbe Interaction group
>			Research School of Biological Sciences
>			Australian National University
>
>	Email:		jjw at rsbs0.anu.edu.oz
>	Phone:		61 6 2495051
>	Fax:		61 6 2490754		
>	Snail:		PO Box 475, Canberra, ACT 2601, AUSTRALIA
>

If you're isolating polyA+ RNA, I would like to bring to your attention a
useful but largely unknown paper:

Bantle, Maxwell and Hahn (1976) Specificity of oligo (dT)-cellulose chrom-
atography in the isolation of polyadenylated RNA. ANAL. BIOCHEM.
72:413-427.

This paper makes several useful observations:

1) RNA can be lost due to non-specific binding to oligo-dT columns. This
non-specific binding can be eliminated by pre-empting the column with
E.coli DNA.

2) Much of the RNA in "poly-A+" RNA preparations is not really polyA+
RNA at all, but rather rRNA that is complexed with mRNA. The authors
demonstrate that this rRNA contamination can be removed by the following
steps:
       a) adsorb RNA onto oligo-dT column
       b) elute RNA from column
       c) Heat at 55C in the presence of 80% DMSO 0.1 M LiCL
          to break up aggregates.
       d) Dilute and re-run on oligo-dT column

This technique works reasonably well. Also, make sure you use reagents
and glassware treated with DEPC.
 
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