Plate setup for qRT-PCR - Too many samples for one plate... (Feb/11/2010 )

Hi to everyone,

I have yet another problem with my qRT-PCR I have 30 samples equally divided into 3 groups (control (n=10), treatment A (n=10) and treatment B (n=10)). I need to check out expression of 3 different genes, one of them is a reference gene. All my samples will be run in triplicate and here is my dilemma how to setup a 96 -well plate.

I see three options how to run this:
1. run one gene in all samples - one gene/plate
2. run all three genes in one group - one group/plate
3. run all samples and all three genes - one replication/plate

What is the best solution here?

One replication per plate provides the best opportunity to compare expression between your reference and test genes. Variations in cycling conditions, room temperature, etc. affect all your samples and the reference equally.