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quantitation of mRNAs-best way?

Tracy Aquilla aquilla at salus.med.uvm.edu
Tue Jul 26 16:01:23 EST 1994


Hi Bionetters,
    I hope someone can help me here. I have been trying to quantitate mRNAs
(steady-state levels) for several cardiac proteins for the past several
months, using primary cultures of neonatal rat cardiomyocytes. Up to this
point, I have been running Northern gels of total RNA, blotting them, and
then hybridizing to various oligonucleotide probes and quantitating the
bands using a Bio-Rad phosphorimager. I am wondering how other people
quantitate mRNAs, and whether the approach I am using has been used by
anyone else. I am having a few problems with this method, particularly in
detecting certain mRNAs which are expressed at low levels, in accurately
quantitating bands which migrate very close to the rRNA bands, and in
getting good signals from the membranes after multiple probing and stripping
cycles. Lately, I have been considering changing to a RNAse protection
assay. Does anyone have any suggestions? Feel free to post here or email
directly to me. Any help would be sincerely appreciated. Thanks.
    Tracy



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