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Replicates

Replicates - Real-Time PCR and Quantitative PCR Forum

Replicates - Real-Time PCR and Quantitative PCR Forum


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  #1  
Old 01-04-2010, 02:29 PM
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Default Replicates



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!
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Old 01-25-2010, 10:35 AM
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Default Re: Replicates

Hi,

I am not familiar with the Pfaffl method, but seems to me its best not to pool the samples - the average value of 6 samples is much more informative than the one value of a pooled sample. however as it is a lot of work, and to save reagents, you might want to pool every two (or even three) samples .
considering the second question - think you should usually repeat the experiment. but hard to answer without knowing the experimental design.
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