jjw@rsbs0.anu.edu.au (05/28/91)
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). The person concerned doesn't have a way of monitoring A260 of washings from an oligo dT column directly (the monitor is broken and the lab has no money to fix it), and would rather not have to take aliqots to a spec to read A260. She has tried oligo dT paper, but is really after something that will provide a greater final quantity of clean PolyA+ RNA. I've suggested the (Pharmacia?) kit in which the oligo dT is coupled to magnetic beads, but this is an expensive kit and my friends lab isn't rich. Are there any (relatively cheap), simple protocols out there that will give large amounts (>25ug) of PolyA+ RNA. Please pass any hints/suggestions/protocols on to me and I'll pass them on to my friend. (I'll be quite interested in the answers myself, anyway!) Thanking you all in advance, ******************************************************************************** Jeremy Weinman Plant Microbe Interaction group Research School of Biological Sciences Australian National University Email: jjw@rsbs0.anu.edu.oz Phone: 61 6 2495051 Fax: 61 6 2490754 Snail: PO Box 475, Canberra, ACT 2601, AUSTRALIA ********************************************************************************
frist@ccu.umanitoba.ca (05/28/91)
In article <1991May28.165855.1@rsbs0.anu.edu.au> jjw@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@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. =============================================================================== 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@ccu.umanitoba.ca | Office phone: 204-474-6085 | - Robert A. Heinlein FAX: 204-275-5128 | ===============================================================================