DeltaDelta Ct Relative or Comparative Quantitation - methods of relative quantitation for qPCR determining gene expression (Nov/09/2008 )

hello!

I'm an hounours student validating some microarrays with a RT-qPCR, and was wondering which method to use to quantify my results.

When I say quantify, I mean relatively. I don't need the actual number of molecules of DNA, I just need to be able to compare the control gene expression levels with the target genes expression levels in a meaningful way.

I'm running my PCRs on a RotorGene 6000 (Corbett Life Science), and it gives 2 options for relative quantitation; the deltadelta Ct method, and the comparative method.

The delta method normalises the target results in relation to the control results and a calibrator, the other method only requires a calibrator.

I guess my question is - which form of analysis should I use, and will one be considered better than the other?

I'm thinking "D'oh! the delta method is better, it accounts for more, but maybe I'm just complicating things?

Would appreciate anyone's opinions.

Cheers

-Tamm-

QUOTE (Tamm @ Nov 10 2008, 04:56 AM)

I guess my question is - which form of analysis should I use, and will one be considered better than the other?

I'm thinking "D'oh! the delta method is better, it accounts for more, but maybe I'm just complicating things?

The delta-delta is golden standard in relative quantification.
But I don't understand what you call "control results" and what a "calibrator". Delta-delta uses set of samples, one of which is called calibrator or control sample, and others are compared to it.
Then, on each sample and calibrator, you run a PCR for gene(s) of your interest and gene(s) called the reference, which is normalised to. These are usually housekeeping genes, that don't change expression between the samples. You can also use genes that showed no difference on the microarray as reference.

Then you have (Ct target - Ct reference) for calibrator (first delta) minus (Ct target - Ct reference) for each sample. That's the second delta.
IMHO the comparative method doesn't use reference, but for further details refer to the user manual.

-Trof-

thanks for that!

i guess i confused myself, but now i get it - my supervisor wants me to do as little work as possible because i'm running out of time for my project, but if delta Ct method is the golden standard, then that's the one i'll be using.

thanks again,

tamm.

-Tamm-

I've read the user manual for the Rotorgene 6000 a million times (which is why I'm confused), and it offers "Comparative Quantitation" which uses a 'calibrator', and the "Delta Ct Quantitation", which requires a validation experiment prior to running the PCRs on the target and reference genes.

The calibrator in the "Comparative Quantitation" is a reference sample (housekeeping gene), that you choose manually once you've run a pcr.

This validation step for the "Delta Ct Quantitation" requires I do pcr on serial dilutions of the target and reference genes' DNA, to compare the reaction efficiencies of the PCR for each one (as the Delta Ct method assumes the reactions efficiencies are almost equal, denoted arbitrarily by the number 1). This would also pinpoint any errors generated during the pipetting of the reactions.

I thought that the Delta Ct method would account for more, by encompassing the the reaction efficiencies, I just wanted to clarify that it was the best way to go...

Did you know you can also use a relative std. curve? A paper by Applied Biosystems (Bulletin #2, Relative Quantitation of Gene Expression) compared some different methods... I think I've read too much.

http://www3.appliedbiosystems.com/cms/grou.../cms_040980.pdf

-Tamm-