I am doing real time PCR experiments and I am having problems with reproducibility fo the results. I recently switched from Syber Green to Taqman and the first experiment was total rubbish- I am using delta-delta Ct method for calculation.

Any suggestion on how to calculate the results accurately? and how to optimise the cDNA concentration and conditions:-)

DNA concentrations can be determined spectrophotometrically, but it's cumbersome and not very accurate. I had this problem during my PhD, but I stuck with the SYBR-green. Fold-differences in starting template concentration between any two samples can be found by using:

2[to the power of](Ct2-Ct1).

PCR is exponential, not linear, hence the 2[to the power of].

Hope this helps. (up)

Sana, I performed serial dilutions of cDNA for each of my primer sets, this gives you a standard curve that performs several functions:

1) Identifies the optimal cDNA dilution (aim to get a suitable Ct with all primers)
2) Enables you to calculate primer efficiency (not everyone uses this in their calculations but it's a good idea)
3) Provides you with a standard curve which you can use to determine a suitable threshold, if you wish.

I stopped using delta-delta-Ct and started using the REST program as I prefer it, horses for courses though, there's no one right way. If you're only using a single HKG it's not such an issue.

In terms of assessing the cause of poor reproducibility, have you checked your machine? I've found significant differences across plates, especially between the inner and outer wells on older machines using a solid heating block. If you want to assess this, make up a mastermix and cDNA solution sufficient for the entire plate, pipette it out and see what the well-to-well variation is like. Also, repeating any given experiment will give you a figure for the inter-assay deviation which is the other concern.

Forgot to say, if you haven't already found it, this site http://www.gene-quantification.info/ has an absolute mass of information and links, I use it all the time :-)