Anyone successfully doing RNAse protection Assays?

Tracy Aquilla aquilla at salus.med.uvm.edu
Wed Mar 6 11:04:06 EST 1996


In Article <1996Mar2.174223.8216 at mcrcr6>, vilimf01 at mcrcr6.med.nyu.edu wrote:
>In article <3135F21D.DA0 at unixg.ubc.ca>, genes at unixg.ubc.ca writes:
[snip]
>     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

That should tell you something! ;-)

>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

Nothing personal, but I think your lack of success using this system
probably has more to do with your technique than the kit itself, unless you
somehow got a 'bad' kit (very unlikely). I've been doing RPAs successfully
for a few years now (almost every week), using Ambion's RPAII and Hyb-Speed
RPA kits. It's certainly not a forgiving technique and demands the utmost
care and attention to detail, particularly in the elimination of RNAses (or
more specifically, avoidance of contamination) and the preparation of clean
RNA. However, I've had whopping success using both kits, although the RPAII
seems to be 'easier' and more forgiving, while the Hyb-Speed RPA is faster
and more sensitive. I've been able to detect messages of "intermediate"
abundance from fetal and neonatal tissues which have very low levels of
expression, and routinely use as many as five different probes in a single
assay on 2 micrograms of total RNA. My images usually have no background and
nice sharp, BLACK bands. The key is really in making the probes. The trick
is to get clean, RNAse-free probes of the proper specific activity. It's
essential to do all the controls you can think of when you first try this
technique. I highly recommend the RPAII: it's a nice fast single-tube RPA
and it's not so bad once you get it all figured out! However, don't expect
it to work that well the first time or two unless you're an experienced
veteran with RNA.
    Tracy



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