real time PCR (using Sybr green) - (Sep/23/2004 )
I am having problems with 2 things.
Firstly, we have difficulty normalizing our cDNA concentrations...any advice...UV specing doesn't seem the best...
Also, with real time usually I don't get primer dimers but now as the DNA concentration seems low it is a problem.
I have done a "gradient' to determine the best annealing temp but this is still a problem and I can't seem to sort it out. I'd be grateful for advice. I tried to increase the magnesium chloride conc. but that caused the efficiency to be very high.
Also is it possible to graph the fold change as an individual value in terms of expression and not in comparison to a control?
Any advice would be much appreciated.
one way to check/normalize the cDNA synthesis is to look at an 18s rRNA assay from 1:1000 or 1:10000 diluted total RNA. You can also do scintillation counting on TCA precipitated cDNA if you include P32-dCTP in the cDNA synthesis.
Regarding specificity and primer-dimer, well, i dont have a good answer. Perhaps you can use less primer when you have less sample to reduce primer dimer, but don't add more Mg, if you allready have trouble with specificity.
regarding teh last point "Also is it possible to graph the fold change as an individual value in terms of expression and not in comparison to a control?"
I would say no: real time PCR is a relative measurement, not an absolute.
Søren M. Echwald, MSc., Ph.D.
One Real-time PCR kit, which covers 38.565 genes