Comparing runs - is it possible without a standard? (Dec/15/2005 )
I am doing some species identification work, using QPCR because it is more sensitive that conventional PCR, we don't have a positive control to run alongside and I was wandering if it would be possible to compare Ct values from different runs if the same amount of DNA is added to every reaction and the threshold is set the same on the different runs?
um, not so much. sorry
your housekeeper is not a positive control; it is a value to use, to normalize against tiny errors. and I do mean very tiny errors...it is very sensitive technology
u can use linREG (ramakers 2003) to generate relative expression values without using a standard curve
How can u not have a positive control? surely you need something to have optimised your assay with??
Regards
you could perform a Q-PCR run and then clone the subsequent PCR reaction into the TOPO vector. You will then have cloned the amplicon you are looking for which can then be used as a positive control
someone in my neighbouring lab once did that..... no positive controls in Q-PCR i mean.... they had a HELL of a time trying to publish it and ended up re doing it all with one in the end.... so be aware of that possibility.
good luck!