RNase Protection Assay-HELP!

John Morlan jmorlan at SFSU.EDU
Wed May 14 13:15:15 EST 1997


Vivanova,

I too have been doing non-radioactive RPA assays for awhile.  Some
questions for you:

a)  You say that you have low efficiency of electrotransfer.  How do you
know this?  Have you eliminated other possibilities?  What does your
autorad look like?

I have performed successful transfers under the following conditions:

0.5x TBE (chilled)
1 hr @ 200mA or 1/2 hr @ 400mA
using a Hoeffer TE 70 or TE 40 electrotransfer unit.

b) Why are you staining in EtBr?  Is this to visualize your bands for probe
purification?  If so, are you extracting in butanol to remove EtBr?
Alternatively, you can avoid EtBr by using UV shadowing to visualize your
bands.

c) Regarding your probe and beta-actin, when you say you see the same band
pattern, it sounds like your titin and actin probe are the same size!  Not
a good idea, especially if you are going to run them in the same lane.
Your different probes need to be different sizes so you can tell them apart
on your autorad.  Alternatively, you may not be running your gel long
enough to see a difference in size.  What are your gel dimensions, and how
long do you run the gel?

d) If you are seeing exactly the same thing in every lane, you may be
seeing an artifact of your DNA template.  To alleviate this problem, make
sure of the following:

1. DO NOT treat your transcription rxn w/ DNAse prior to gel-purification
on PAGE/urea.

2. Make sure your DNA template for probe transcription DOES NOT have a
3'-overhang.  Non-isotopic autorads are notoriously sensitive to
artifactual transcription of the opposite strand.

If I did not understand your original problem, the above may not make any
sense.  Hope it helps, though.

John Morlan
Department of Biology
San Francisco State University
1600 Holloway Ave
San Francisco, CA 94132

(415) 338-7099

INTERNET: jmorlan at orion.sfsu.edu



>Hello, everybody!
>
>Does anyone have any experience with RNase Protection Assay? I have been
>doing it for a few months now, using a non-radioactive method for
>detection (biotin) and I have problems with transferring from the PAGE
>to a nylon membrane (low efficiency of transfer). My other problem is
>that after I stain the urea/PAGE with EtBr I see the same band pattern
>with my control probe (beta-actin) and with my probe (in this case for
>titin). It's absolutely the same picture-with actin, with titin and with
>both probes. What I am looking for is differential expression of titin
>at different stages of primary chick embryo muscle culture and if there
>is no way of comparing titin's expression with that of actin
>(housekeeping gene), I am in a bad shape. I would greatly appreciate any help!
>
>Thanks a lot in advance!
>
>
>Vesselina Ivanova
>
>Graduate student in the Department
>of Biochemistry and Biophysics
>Iowa State University
>--
>Vesselina Ivanova
>vivanova at iastate.edu





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