Anyone successfully doing RNAse protection Assays?

vilimf01 at mcrcr6.med.nyu.edu vilimf01 at mcrcr6.med.nyu.edu
Sat Mar 2 16:42:23 EST 1996


In article <3135F21D.DA0 at unixg.ubc.ca>, genes at unixg.ubc.ca writes:
> I have started doing RNAse protection assays using an Ambion kit(RNAse 
> II). I can't seem to get it to work. The probe is 600 bp and is made 
> from a pGEM plasmid containing an insert. The target is made from a 
> longer sequence that is in another plasmid (probe was actually 
> subcloned from this longer sequnce). I am trying to titre the reaction 
> so as not to use up my total RNA sample that is limited. The control 
> for the kit works fine. Can anyone give me some pointers on making the 
> probe or the target sample and getting this to work. 
> Any help will be greatly appreciated.
> Craig

     I too have used the RPAII kit for doing protections.  I had no
sucess in detecting my message with the kit.  I even had problems
detecting the sense RNA control that I ran off my gene of interest. (I
highly recommend this control for troubleshooting RPA).  Other lab
members also reported problems with the RPA II kit and recommended the
old RPAI kit which is basically the procerure from current protocols
in molecular biology.  The control sense RNA now gave a HUGE signal
and, after gel purifying the probe (also highly recommended), I
managed to detect my message in 20ug of total RNA.  In speaking to the
Ambion people, they confirmed that the new RPA kit is less sensitive
than the old procedure.  The phenol step is somewhat cumbersome, but
getting the result is worth it.  For rare messages, or if RPAII isn't
working, I recommend the old procedure.  By the by, I also modified
the hybridization step as per Mironov et al NAR 23(16): 3359 using a
buffer devoid of formamide and hybridizing at 85 C for 2-4hr.  Faster
hyb with better results in my hands, and my probes were 650 and 300bp.
Hope my experiences are helpful, good luck.

			Usual Disclaimers		SVEN



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