Plate setup- have I covered everything? - help- qRT-PCR newbie in state of confusion! (Jun/01/2006 )

Hi there everybody
I've been busy reading up about lots of aspects of qRT-PCR but I'm having problems with getting my head around how I'm actually going to set up my plates. I'm going to do a 2 step PCR and will be using 96 well plates.

Here is my experiment briefly:
2 conditions, 6 samples in each condition- so 12 RNA extractions followed by 12 cDNA synthesis.

I was going to set up my plate thus:
each of my samples in triplicate= 36 wells
one no RT control for each cDNA synthesis= 12 wells
3 blanks= 3 wells
3 standards (to allow me to normalise over plates-not sure what I'm going to use for this- any suggestions?)= 3 wells

so far that's 54 wells but what do I do about my reference genes?
I was going to do a relative quantification against 2 reference genes so is that a further 2x36 wells? Am I supposed to have my reference genes on the same plate?

I'm very confused so any advice would be much appreciated!
Thanks
Alison

have you actually run a plate yet? (I'm not picking on you if that sounded testy; just need to know where to start )

so first, are you using ddCt or standard curve? if you are using standard curve, you need to allow for a standard curve on each plate as well.

I don't understand your 3 'reference' wells. what system are you using?

I would ask if you have read ABI's "user bulletin #2"? even if you don't use their stuff, they give good information on getting started and how to set up. I think maybe backing up just a little would be good right now, so you don't waste your samples. initially, I would say you need to run your two conditions on separate plates in order to fit it all and have all your controls done properly. also, there are tips in ABI's manual about the proper way to set up RQ studies.

good luck!!!!!

Hi there
Thanks for the advice. I'm planning on doing relative quantification and using LinReg to determine well to well efficiencies. I actually meant a calibrator when I described the standards wells. I think I'll probably do all the minus RTs on one plate and each ref gene on a separate plate (if my interplate variability is low enough). That would leave me just samples, blanks and the calibrator on each plate. (I think!).
Thanks
Alison