Standard curve

[mvermehren]

I found this explanation: "Prepare five (5) ten-fold serial dilutions of cDNA template known to express the gene of interest in high abundance. Use each serial dilution in separate real-time reactions, and determine their threshold cycle values. In a base-10 semi-logarithmic graph, plot the threshold cycle versus the dilution factor and fit the data to a straight line. This plot is then used as a standard or calibration curve for extrapolating relative expression level information for the same gene of interest in unknown experimental samples. The relative quantification calibration curve result for the gene of interest is normalized to a that of a housekeeping gene in the same sample, and then the normalized numbers are compared between samples to get a fold change in expression. A standard or calibration curve must be generated separately for each gene of interest and each housekeeping gene." My question: Do I have to perform a new standard curve with each and every pair of primers on every plate setup or is one standard curve sufficient for each pair of primers regardless of how often I use these primers in different pcrs? I would not be able to do the latter, this would surpass the amount of available wells on my plate setup - makes no sense? Thanx for help. Margaret

[danfive]

Every pcr plate needs a standard curve, for target gene(s) and housekeeping gene---are you doing duplex PCR?

[mvermehren]

No I am not doing duplex PCRs. I don't want to do absolute quantification, only need the efficiency (slope) of the primers. Can't I run them separately? My PCR conditions always stay the same.
Thanx for help, Margaret

[danfive]

Originally Posted by mvermehren

No I am not doing duplex PCRs. I don't want to do absolute quantification, only need the efficiency (slope) of the primers. Can't I run them separately? My PCR conditions always stay the same.
Thanx for help, Margaret

Hi, you need to run a standard curve for each primer set. They will vary from one PCR to another (interassay variation) and they are a very good quality control. I understand that you are normalizing to PCR efficiency, this is very nice way to do it. Good luck.

[danfive]

These were my references for some PCR normalization work I did two years back, I believe I used the Pfaffl method. Good luck.

http://www.embl.de/courses/qPCR/2007...l-NAR01e45.pdf

http://www.embl.de/courses/qPCR/2007...mal%20GB02.pdf

http://www.umich.edu/~caparray/Files...ddCt_paper.pdf