PCR efficiency for qRT-PCR analysis - PCR efficiency for qRT-PCR analysis (Jan/10/2008 )

I have an endogenous control gene and about 5 target genes I would like
to try initially. For various types of analysis (e.g.delta delta CT), I
need to calculate and have close to 100% PCR efficiency for the qPCRs
for all the genes. I have found that I am not about to check this very
well as I am not able to generate good standard curves with my 'test
template' which a cDNA generated from the same tissue type I want to use
for my experiments. I think this is because my cDNA only has low amounts
of the target templates.My endogenous control PCRs are Ok but some of my
target genes are present in relatively low quantities and so it makes
determining efficiency difficult. I can see this approximately in normal
PCRs, too.
For a couple, I have done a rough trial using plasmids containing the
cDNA target sequences and those qPCRs give better curves. Is it OK to
calculate efficiencies using plasmid DNA? I guess the fact that my cDNAs
may have very low quantities of target material could be a problem too
but I guess I need to see the PCRs being equivalent first.
Would anyone have some advice?
Thanks.

hmmm

the amount does not necessarily have anything to do with the efficiency of the reaction. what I mean is, your HKG can have a much higher amplification than your GOI and it's still OK, as long as their efficiencies are close to 1.

have you read ABI's User Bulletin #2, from their website? it's a bit laborious to read, but if you set up your control plates and follow the algorithms outlined in this publication, you will get what you need in terms of optimizing efficiency of your reactions.

good luck

Thank you for your reply. I really appreciate it. I read the ABI user Bulletin and it is most helpful. I think I understand the concept of comparing relative efficiencies better now.
However, I still have the question of whether it is OK to use a dilution series of plasmid DNA to determine the efficiency of target gene amplification in the case of very low amplification of the target gene with cDNA generated from the RNA tissues of interest.
This could be the case where a gene is hardly expressed/not expressed in the tissue of interest but I still want to include that target gene in the study.
If the expression is so low that I can't generate a linear curve from a dilution series because my undiluted cDNA generates almost no product or no product at all, then can I use plasmid DNA as template for the relative curve to show that the PCR primers are not the reason for no product.
In the ABI User Bulletin #2 example, the relative standard curve was generated using a tissue culture and the samples were various organ tissues. Using this as an example, would it have been OK to substitute a plasmid containing the cloned cDNA of the gene if even the tissue culture cDNA did not generate a product/or very little product for a gene of interest?

Hi,
Well, if you are doing relative quantitation, you are supposed to do a std curve using RNA (or cDNA). But since you would like to do delta delta Ct and the std curves are only to get the efficiency, it should be ok. If you do absolute, then your sample Cts must be to the left of your std curve, i e, you std curve must have dilutions more dilute than your samples for quantitation to be accurate.
But I don't understand if you don't expect the gene to be expressed, why do you want to quantify it?

Cheers,
Chris