Interpretation of comparative quantitation result - (Mar/22/2007 )
Hi friends...need help here..
It's been about a month since i've started my realtime exp. I've managed to prepare standard curves with good efficiency (90-110%) for both my GOI and HKG, using plasmid and gDNA. Previously facing the problem of gDNA contamination in cDNA (for gene expression studies), but thank goodness, the problem has been solved.
Through a discussion with a friend of mine who's experienced in qPCR, we can use delta delta Ct method to quantify gene expression level, without the need to prepare the standard curve. In that case, we assume that the efficiency is 100%. So that means the reason we prepare the standards is just to make sure that our primer and reaction efficiency doesn't change with varying template concentration. Am i right so far?
Now coming to the actual problem. I've run my comparative quantification, and planning to use the delta delta Ct method to calculate fold change in expression level. Got the results, but seems a bit weird. I used non treated control as the calibrator, and the value in the relative quantity chart for calibrator should be 1, right? What does it mean if the our unknowns got a value below 1? It means that the gene is down regulated somewhat. Am i right so far? For comparative quant, the software didn't display any dissociation curve. Why is that?
Now i'm not sure whether the results are valid or i should repeat them all over again? Or is it mean that the expression is too low somehow? Hope anybody can help me sort this thing out...
um, I can answer part of that, anyways!
yeah, if you set your HKG at 1, if you get <1 it's due to reduction in expression. alternatively, you can think of it like a percentage; 1 is equivalent to 100%, so 0.65 is 65% of the expression level seen with the calibrator/control sample. likewise, if you get a result of 3.4, you know that it's 340% of the expression level seen in the control
as far as getting your software to show diss. curves, I can't help you there?? which software? which machine? do you have the manual? I'm guessing there was a setting somewhere that you had to choose?
Tq aimikins.. u helped me understand better..anyway i'm using Mx3000P machine from stratagene. Must be a way to set the dissociation curve right? And for the comparative quant, i guess i want to repeat to see the expression of all the cDNAs, whether it is overexpress or the opposite. Thanks a lot..
Are you using Taqman or SYBRgreen? I am not sure, but as far as I know you cannot get a dissociation curve with taqman...