RNase- or S1 nuclease Protection Assay & ABI377

Carla Carla
Mon Sep 1 12:58:59 EST 1997


Arthur, Osterop, Ph.D., osterop at tch.fgg.eur.nl wrote:
> 
> Does anyone have experience with or is currently trying to set up RNase-
> or S1 nuclease Protection Assays using the ABI377 sequencer? I am
> considering this application as the probe is stable in contrast to the
> radioactive versions of the protection assays. However, I don't have any
> experience with protection assays, thus I may be ignorant of very
> obvious pitfalls. What could be possible problems when switching to
> fluorescent probes? Does RNA polymerase incorporate fluorescent
> nucleotides? Is the fluorescent probe/RNA complex resistent to RNase or
> S1 nuclease?
> Thanks in advance for thinking with me.
> With best regards,
> 
> A.Osterop Ph.D.
> Erasmus University Rotterdam
> The Netherlands
> osterop at tch.fgg.eur.nl
I would also be interested in hearing suggestions about this intriguing
idea.  Our lab has been trying to find a good way to quantitate a rare,
high GC RNA for years.  RPA has been somewhat successful, but the
quantities of RNA needed and the labor intensive needs of this
methodology are daunting.  To automate this would be great.  The
alternatives we have considered, such as automating quantitative PCR on
a 377 or 373 or purchase of the ABI Quantitative PCR system have been
haunting us for a year.  Does anyone believe the probes for RPA could be
sensitive enough give a signal with a rare transcript?

Carla Drebing
Univ of CO Health Sciences Center
Denver CO
Carla.Drebing at UCHSC.edu



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