Ribonuclease Protection Assay Controls ?

Tracy Aquilla aquilla at salus.med.uvm.edu
Mon Dec 19 09:06:33 EST 1994


In Article <3cu257$qgu at apakabar.cc.columbia.edu>,
jjm3 at sawasdee.cc.columbia.edu (Jeffrey Joseph Martino) wrote:
>OK here it goes: Doing Ribonuclease Protection Assays on total RNA
>                 from various mouse cells/tissues (Northern no signal,
>                 and initial RPAs - no signal)
>
>  - actin controls give a good signal.  
>
>Actin isn't too rare.  Want to put a detection limit number on that
>RPA negative result.
>
>I thought adding known quantities of an RNA back to sample as a control
>would be OK to check detection limit, but I promised to keep an eye out
>for an endogenous alternative:
>
>  Any ideas for a good (not tissue-specific) low level message (with
>  probes available) for a control?
>
>
>Thanks for any and all comments.
>
>

To determine the sensitivity of your assay you could try reducing the amount
of total RNA added to the hybridization until you reach the limit of
detection for your internal standard. Even if the mRNA is abundant, this
should work. Alternatively, try adding more total RNA (up to 50 ug) to the
hybridization for detection of the rare message you are interested in. Good
luck.
    Tracy



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