Better methods for PolyA+ please

frist at frist at
Tue May 28 10:04:39 EST 1991

In article <1991May28.165855.1 at> jjw at 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
{ ... details of request deleted}
>Jeremy Weinman		Plant Microbe Interaction group
>			Research School of Biological Sciences
>			Australian National University
>	Email:		jjw at
>	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.

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
       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.
Brian Fristensky                |  
Department of Plant Science     | Freedom begins when you tell Mrs. Grundy
University of Manitoba          | to go fly a kite.
Winnipeg, MB R3T 2N2  CANADA    |  
frist at          | 
Office phone:   204-474-6085    |   - Robert A. Heinlein
FAX:            204-275-5128    |

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