RNA slot-blot

[eric anderson]

In article
<Pine.A41.3.95.970129170835.78290F-100000 at green.weeg.uiowa.edu>, "A.
Kumar" <akumar at blue.weeg.uiowa.edu> wrote:

> Is the slot-methods reliable for quantitative RNA analysis ? We used a
> slot blot with different amounts of the same RNA and probed it with a cDNA
> probe. The signal as measured by densitometry was proportional to the
> amount of RNA but the slope was not 1. I am concerned of the reliability
> of this method Vs conventional Northern blot analysis. The protocol
> followed was as mentioned in "Current Protocols" (the red book). If there
> is a better protocol, I'd appreciate any input.
> Thanks.
> Ashish Kumar

if you go to page 4.9.12 in the Red Book, the commentary section for
Northern and Slot-Blot analysis of RNA, you will find the following
quotation:
"Although easy to perform, RNA slot-blot hybridization is one of the most
problematic techniques in molecular biology."

they go on for 2 long paragraphs explaining the pitfalls of quantitative
slot-blotting.  this was one major reason that we switched to Northerns
for a final analysis in a differential expression screen.  about 2/3 of
the clones that were "upregulated" in the slot blot turned out to be
unchanged or downregulated when examined by Northern.

so my suggestion is to go with Northerns if you want an absolute quantitation.

good luck,

eric

-- 
Eric C. Anderson
Memorial Sloan-Kettering Cancer Center
Sloan-Kettering Institute
1275 York Ave. Box 470
New York, NY  10021
(212) 639-2977
e-anderson at ski.mskcc.org