Standard Curve vs DDCT

[Brian P Higgins]

What are you running your standard with?  I would just clone your PCR
products, then use the miniprep to run your standard curve--you'll never run
out of standard.

Some are fans of of the ddCt method.  There are some very detailed versions
of the ddCt method (Pfall comes to mind, along with another version called
REST), but I simply don't trust it.  There's a recent review by Bustin
(within the past couple years) that addresses the issue of quantitating RNA
via qPCR.  The basic problem with the ddCt method is that it assumes equal
efficiencies among all your primer pairs.  There may be ways to take this
into account now, but I have always treated each primer pair separately,
then done the math after the fact.

Regardless of the method you choose for quantitation, I would run a standard
curve for each primer pair--I think Bustin points out in his review that a
small difference in PCR efficiency can lead to gross miscalculations.  To
run the standards, just clone your PCR products and run serial dilutions of
the plasmid.

good luck
brian

On Fri, May 16, 2008 at 12:56 PM, Yvonne Couch <yvonne.couch from dpag.ox.ac.uk>
wrote:

> Hi guys,
>
> I've recently had some problems getting RNA for some qPCR but my main
> problem is that I run a standard curve with all my sets of primers to make
> sure I can compare the efficiency of the reaction properly.  With my
> current
> RNA yield I don't have enough to run a standard curve.  There are
> differences of opinion in our lab, one person says always run a standard
> curve to make sure the efficiency is the comparable and one person says you
> don't need a standard curve just use the controls to compare levels and do
> Delta:Delta CT.
>
> Any thoughts?
>
> Cheers
>
> Yvonne
>
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