In article <49l20s$bq at mark.ucdavis.edu>,
ez008413 at bullwinkle.ucdavis.edu says...
>>I'm about to start gene expression studies at the transcription level but
>I have not decided between RT-PCR and Northern analysis as the method
>use. I only need to have a yes or no answer for expression initially and
>then have some idea on the level of expression. How do the methods
>compare in terms of ease/difficulty in running the experiments, and in
>terms of amount and quality of information?
>>Thanks for any advice.
I agree RT-PCR is faster and definitely requires much less RNA. I would
recommend using Gibco's Trizol (for total RNA) and their Superscript Kit
--you can have results in a day. No stupid blots and probes and hybs.
No isotopes--ethidium staining should be enough. You prob. should
sequence the product band though just to be sure it's the right thing. Just
be SURE to run a RT minus control--omit the reverse transcriptase to make
sure you have no genomic DNA. We had LOTS of trouble with that using
other mRNA purification methods--the genomic DNA seemed to stick to
ANY incarnation of oligo dT cellulose. The cells were tetraploid so there
was extra DNA. The gene also had no introns so the products were the
same size. We found that a double extraction of the DNA with trizol gave
us consistently clean RNA.
For quantification, I used the Clontech kit, but it was very flaky.
Specifically, we would find that the concentration ranges for the same pool
of RNA were different each time. It seemed like the concentrations of the
competing primers may have changed on storage or something. We would
get strange dilution errors that were impossible to track down. Email me if
you need any further explaination of this if you want more info.
Jody K. Hirsh
Northwestern University, Chicago, IL. USA
jkh141 at nwu.edu