Help: Relative Standard Curve Method - (Oct/16/2007 )
I am needing to do relative QRT-PCR using standard curves. I'm reading alot about diluting a known amount of cDNA for use as a standard. What exactlty is the standard? Is this cDNA separate from the experimental samples? Or can the same samples used for the calibrator be used as the standard for the standard curve?
I have done some relative quantifications by qRT-PCR (Ct method) since this method eliminates the need for standard curves (as it's needed for absolute quantification). I prepared a standard curve (known serial dilutions of my cDNA) only the first time to see whether the dynamic range of my normalizer (endogenous control) was similar to the genes I wanted to study.
I guess the trick to standardise your results is always to use the same amount of RNA (your sample) by measuring it before doing the RT, and use the same batch of cDNA (same template) for the normalizer and your genes of interest.