qPCR Standards

[Yvonne Couch]

Hi all,

I appreciate I always seem to ask relatively stupid questions of you all but
nobody in my lab seems to answer simple questions with simple answers!  I
have to run a qPCR on Monday for the first time and I'm not entirely sure
what to do.  I have some cells in which I have knocked down a gene with 3
different siRNA's plus GAPDH siRNA as a control and I have done this at 3
different time points.  So for example siRNA number 1 was applied to the
cells in triplicate and RNA was extracted at 4, 24 and 48 hours, giving me 9
different samples (plus 3 with no siRNA in as another control).  This was
done for all the siRNA's so my total sample number is 48 (12 of which have
no siRNA in).  I have made cDNA for everything but that's where I come
unglued!  I know how to pipette each sample but I'm confused about how to
make standards.  Lots of websites say about pooling cDNA but from which
samples??  And how much do I pool?  And how will this give me a useful
standard?

I'm sorry I sound really silly but I want to know how to do this by
principle rather than by rote if you see what I mean!

Thanks!

Yvonne