Lower probe concentration - lower CT values - (Jan/09/2006 )

Hi everyone,

I am designing an assay to detect viruses in clinical samples using real time PCR. I have found that decreasing my probe concentration (to 20nM) decreases my CT values, therefore making the assay more sensitive (>1 log). However, this results in a much lower normal fluorescence value. Looking at the raw data the fluorescence hardly registers, but after using the RotorGene Analysis software I get really good, reproducible data with high efficiency. Is this a problem that the normal fluorescence is so low? All of the papers I read use a higher probe concentration.

Cheers,

Hordo.

Does no-one know the answer? Or is it just an incredibly stupid question and no-one is bothering to reply?

By the way it is a Taqman assay.

Hordo.

no it's not a dumb question...I just don't know the answer

I do not use Taqman probes, I use SYBR green; I also designed my own system to analyze the data and thus am not familiar with that software

you say your reaction efficiency is really good, those would be the numbers at which I would be looking IMHO

I have seen all sorts of strange things that can happen when you monkey with the proportions in real time, and I have always stayed with the recipe that gave me the best efficiency---but you are looking at viral detection, and I am looking at relative transcript in response to stimuli

anyways, this is why I have not answered your question...

I use TaqMan assays, but have never used a less than 100 nM probe (which also gives me relatively low flourescence, but better efficiency). What instrument are you using? You might contact their tech support and see if low flourescence makes the assay any less valid.

Using a RotorGene 3000.

I have found with any Taqman probes that I use, that if I decrease the concentration to 20nM I get much lower Ct values. I am happy with the assays, but my major concern is whether they will be transferable to other platforms and machines.

Thanks for the replies.

Hordo.