To gel or not to gel
dspinella at chugaibio.com
Mon Jul 7 09:39:55 EST 1997
> Okay, here's the situation. One of my first tasks in graduate
> studies is to isolate mRNA for differential display analysis.
> In trying to familarize myself with the primary literature I have
> gathered that most of the time it goes a little like this:
> 1. isolate and purify mRNA
> 2. RAP-PCR
> 3. electrophoresis of cDNA
> Here's the question:
> Would there be any advantage to performing the electrophoresis
> on the mRNA and isolating differential bands to then be used in
> RAP-PCR? By this I mean, you wouldn't have to use as much
> primer and so forth if you were only amplifying the differentially
> displayed mRNA instead of amplifying ALL of the mRNA?
> Just curious
Well, how is it exactly that you plan to get your differentually
expressed mRNA bands prior to amplification? When you get through with
your literature review and actually electrophorese your RNA, you will
find that there are no bands (differentially expressed or otherwise)
except for the two rRNA bands. There will just be a smear corresponding
to all the various sizes of mRNA in your starting pool. You will also
find that working with RNA is far more difficult than working with DNA
-- it is very labile, doesn't electrophorese into sharply resolved
bands, and requires special electrophoresis conditions (formaldehyde or
glyoxal) which are not amenable to the thin sequencing type gels
required for differential display. I'm afraid you're going to have to
stick with the tried and true approach here.
Nevertheless, its good that you're starting off your graduate studies by
thinking about techniques and "challenging the paradigm". Even though
this particular idea is not a winner, I encourage you to keep thinking
this way. As you get more experienced, you may one day come up with an
idea that revolutionizes the whole field!
Good luck in your graduate career.
-- D.G. Spinella
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