RNase protection probe activities

Richard Rohan rrohan at world.std.com
Thu Aug 10 23:40:36 EST 1995

macdiarm at sbsu1.auckland.ac.nz (Colin MacD) wrote:

>I am setting up an RNAse protection assay and have been having problems with degradation (i.e. radiolytic decay) of my 32P-labelled probes. My probes have specific activities of about 5x10^9 cpm/microgram. I dont get degradation using low sp. activity probes, but need high sensitivity.
>Is 5x10^9 too hot?
>What is the best probe size and activity for high sensitivity?
>I would greatly appreciate any help.

>Dave Whittaker
>whittakr at sbsu1.auckland.ac.nz

We routinely synthesize riboprobes with 800 Ci/mmole 32P-UTP
(approximately 1x10^9 cpm/microgram) using 50 microCi in a 20 microL
synthesis reaction. I don't think that it is possible to use higher
specific activities because of the Km of the bacteriophage RNA
polymerases.  Probe sizes of 200-400 nt usually do quite well.  Probes
larger than 400 nt are more difficult to synthesize and result in more
prematurely terminated products.  Probes that require incorpation of
long runs of U should be avoided (or use another labelled nucleotide).
Gel purification of the radioactive probe helps to reduce background.
This is necessary with longer probes and  when multiplexing probes.
We have not experienced serious problems with radiolytic decay if
probes are used immediately (ie. 2-3 days from time of probe synthesis
or gel purification to electrophoresis of protected fragments).  Using
these conditions we can detect 0.03 pg of a 400 nt target.  This
should be sensitive enough for most purposes unless your RNA is
extremely difficult to come by.

HTH. Contact me if you have further questions.

Rich Rohan
currently unaffiliated
rrohan at world.std.com

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