How can I cofirm differential expression ?
un691cs at genius.embnet.dkfz-heidelberg.de
Tue Dec 17 04:14:36 EST 1996
In article <e-anderson-1612961059330001 at news.ski.mskcc.org>, the honourable
e-anderson at ski.mskcc.org stated:
>In article <32B50A68.765 at unitel.co.kr>, khsw124 <khsw124 at unitel.co.kr> wrote:
>> What should I do next to confirm the differential
>> expression in this case?
>your best bet is to sequence the clones (assuming that you have them) that
>showed differential expression and use that information to make RT-PCR
>primers and do RT-PCR on them. this is a stop-gap measure at best since
>it's not the most definitive assay. i've had differentially displayed
>genes show up by RT and not by northern and the other way around. if you
>absolutely can't do northerns, then go with RT's, but only as a last
I don't agree. Most DD-RT-PCR products have such a low abundance that
you may go nuts trying to show their differential expression by Northern.
Now-a-days, there are a lot of nice quantitative RT-PCR approaches
described in literature. Important thing is to always carry out the
experiments in the linear range, in duplicate, and use Southerns.
An internal standard may do wonders, but is not essential
for each individual RNA tested, as long as the same starting RNAs
May I point you to a classic paper already (*uhm*) which, by extreme
coincedence (*uhm*) was written by us ? (although the references
there drin are probably of more value to you...).
Suter-Crazzolara, Unsicker, Mol Brain Research 41 (1996), 175-182,
"GDNF levels are induced by FGF-2 in rat C6 glioblastoma cells"
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