Hi, I'm new to rt-PCR, and I have a question about replicates. I'll be using the Pfaffl method to compare the gene expression of non-treated and treated fish samples. At each timepoint I'll have 6 non-treated and treated samples. My question is on the number of replicates I should use.
i. Should I pool the RNA from all 6 fish samples or do I consider them as individual samples (separate extractions) and get an average value?
ii. Is an experimental replicate absolutely necessary? As in a repeat of the entire experiment using the same parameters etc?
Thanks in advance!