RNA Slot-blotting woes

Jim jmeador at adnc.com
Sun Jun 9 09:14:27 EST 1996


Gabriel,
I hate to burst your bubble, but you're going about this all wrong. It's
barely accurate to try to quantify an mRNA via a Northern since there are
variablities in binding of the RNA to the membrane, in the percent of
bound RNA that is hybridizable to the probe used, and in the amount of
retention of hybridized probe after the washes. I think the other problems
with simply slot blotting should be obvious. 

IMHO, the only way to accurately measure the levels of mRNA is by using an
RNAse protection assay, which uses solution hybridization. You'll still
have possible variabilities that can underestimate your measurement, but
they are more easily dealt with. In addition, there are better ways to
make standards. Which brings me to the other problem with your approach,
your standard. DNA is "double stranded" which makes it a VERY bad standard
to compare to single stranded RNA. You can never know what percent is
actually hybridizing to your probe, but you can be assured that it will be
rather low as compared to using a single stranded template.

In short, rather than trying to "work out" the problems with your present
line of attack, which will probably consist of getting the numbers to be
what you think they should be, I think you should completely rethink your
procedure. 

Hope this helps, although it isn't what you were looking for,
Jim Meador

In article <4p2e9q$f11 at mark.ucdavis.edu>, ez008413 at dale.ucdavis.edu
(Gabriel Romero) wrote:

> I'm trying to quantify my gene's transcript in several tissues using 
> slot-blotting of total RNA and known amounts of target DNA as standards. 
> My previous northerns show comparable high level expression let's say in 
> tissues a,b and c. When I slot-blotted the same amount of total RNA from
the 3 
> tissues as I ran for the northrens, I got starkly different results: 
> such as, only tissue a showed signal with b and c showing nothing. In short, 
> the northern data and slot-blot data are inconsistent.  Because my northerns 
> have been reproducible, I suspect something is wrong with the 
> slot-blotting. So, in my subsequent attempts, I've turned down the vacuum 
> and also added carrier tRNA to improve RNA retention on 
> the blot. Still no improvement. Could somebody help me troubleshoot?  I'd 
> appreciate any advice.



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