More PolyA+ questions/comments

Marc Lamphier lamphier at po.infosphere.or.jp
Sat Oct 21 10:30:16 EST 1995


To PolyA+ RNA questions (and one product comparison)

1) What is a good indicator of the quality of one's poly A+ RNA prep? 

One person told me that you should look at the 18S and 28S rRNA (in the
case of mouse RNA) that is carried over in most preps and check that the
upper band (28S) is heavier than the lower band (18S). Certainly this is a
good indicator of the quality of total RNA, however I wonder if this is
also true in the case of PolyA+ RNA? I noticed in the technical manual for
Promega PolyATtract mRNA isolation system a photograph of an
ethidium-stained sample of PolyA+ RNA (mouse) and the contaminating 18S
rRNA is in fact heavier, not ligher, than the 28S band. The PolyA+ samples
I have recently isolated (using Invitrogen Fast Track system) also show 18S
staining heavier with methylene blue than 28S  -- and by other criteria the
mRNA looks reasonable: staining of the mRNA is heaviest around 2kb and
extends well up the lane with no obvious accumulation of material near the
bottom of the lane. I wonder if 18S simply binds non-specifically to
oligo-dT better than 28S, and thus more of it is carried over as
contaminant in poly A+ preps than 28S. 

Does anyone know if comparing 28S <-> 18S is a reliable indicator of
quality in the case of PolyA+?

2) How does one probe for total PolyA+ RNA on a Northern blot?

I would like to probe total PolyA+ RNA on my Northerns as a way of
double-checking both the integrity and quantity of the transferred mRNA. I
have seen references to using a 32P-labelled oligo dT probe, however I am
not clear about the conditions. With A::T hybridization using a short oligo
the hybridization conditions must be fairly non-stringent. Promega
literature mentions 50 degrees in 6X SSC containing 1X Denhardt's and 0.1%
SDS. I tried this several times but got no specific signal (I tried a
variety of wash conditions). 

Does anyone have experience with probing for total PolyA+ on Northern
blots?



3) Also for anyone interested, I have used both the PolyATract (Promega)
and Fast Track (Invitrogen) systems and I recommend Fast Track. Yield with
FastTrack has been several-fold higher (i.e. 4 - 5 X!) than with PolyATract
and and fewer problems with degradation (perhaps no problems, depending on
the answer to question #1). Degradation with PolyATract may have been due
to my method of isolating total RNA (guanidinium/phenol -> ethanol
precipitation) prior to using the kit and not due to the kit itself -- I
don't know. The FastTrack protocol is a "one-step" method, i.e. oligo dT is
added directly to lysed cell extract, and thus there are fewer steps where
degradation might occur. 

Marc Lamphier
University of Tokyo
lamphier at po.infosphere.or.jp



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