isolation of PolyA+/A- RNA

N. Salazar nsalazar at U.WASHINGTON.EDU
Wed Jul 23 16:17:43 EST 1997


Hi there,
		I have used the Pharmacia kit to isolate PolyA+ and polyA-
RNA from Plasmodium falciparum and have some questions about what to
expect in terms of recovery.

I started off with 910 micrograms of total RNA, and got 14 micrograms of
PolyA+ RNA, this represents about 1.6%. According to the information
supplied with the kit, one is supposed to get about 2% of PolyA+ RNA.
However my boss told me that that is not true since She got 4% when she
did it. As for the polyA- RNA, I got 400 micrograms that I recovered after
precipitating the first effluent (that is what it did not bind to the
oligodT column). I also recovered polyA- RNA after the three high salt
washes, that was 104 micrograms. Adding up all these RNAs, I get something
like 520 micrograms. My question is where did the remaining 400 micrograms
go ? It seems that I lost them!!. What was wrong with the protocol??

I would like to hear from people who have isolated polyA+/A- RNA to tell
me the yields that they usually get. If there are some good papers for me
to read about it , that would be good too.

			Nelson Salazar
			nsalazar at u.washington.edu







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