kim at m44.unm.edu
kim at m44.unm.edu
Tue Oct 22 16:23:03 EST 1991
I am trying to do Poly(A)+ selection of RNA using either Oligo(dT) cellulose
or Amersham Messenger Affinity Paper. I have been having a lot of trouble with
the procedure, and I need some advice.
I have dry oligo(dT) cellulose, which I prepare for selection by first
hydrating it in binding buffer (Tris/EDTA/SDS/0.5 M NaCl), and then washing
with several bed volumes of 0.1 N NaOH, which is recommended in several
published protocols. I am not entirely sure what this step is supposed to do,
however. If anyone can answer that, I'd be grateful.
After the NaOH, I wash the cellulose with water, and then with Binding
Buffer, restoring the pH to 7.5. The cellulose returns to a white color. I
then heat my RNA sample to 65 C for 10 minutes, and chill on ice. I add 2 x
Binding Buffer, and apply to the column.
The column is washed, after 10 minutes of binding, with 0.5 M NaCl and 0.1 M
NaCl, and then the RNA is eluted with TE buffer heated to 65 C. The eluate is
re-heated and re-applied to the column to squeeze more stuff out of it.
When I run the "Poly(A)+" RNA on a gel, however, there is no attenuation of
the rRNA bands, compared with an equivalent mass of total RNA. Nor is there an
enhancement of these bands in the Poly(A)- fraction. It is as if there is no
specific binding at all. My yields, if that is the correct term, are around 1%
to 3% of the total RNA applied.
I have tried doing this using a batch method, as well. I have also tried
using messenger affinity paper, with the same results. The discussion in
Current Protocols and Maniatis suggests that the rRNA bands should be almost
undetectable in a poly(A)+ fraction.
What kind of mistakes could I be making to prevent specific binding?
Is the NaOH wash step really necessary?
Why do some procedures call for LiCl and some for NaCl?
Why do many procedures call for SDS or Sarkosyl?
I wish to make a cDNA library with this RNA. As long as I am using a
poly(dT) primer, do I really need to fractionate the RNA, or could I simply use
an amount of total RNA in the first strand reaction that would contain an
appropriate amount of poly(A)+?
Am I stupid? clumsy? unlucky? or all three?
Thanks in advance . . . Daniel Kim (KIM at FLOVAX.LANL.GOV)
More information about the Methods