So I'm using qPCR to validate knock down after RNA interference. However as luck would have it, my most interesting gene is giving me the most problems.
Let me just start off by saying that I have optimized these primers as well as I could and due to limited samples my control group samples are already finished, so I can under no circumstances use new primers, nor even repeat the experiment under different conditions as that would require me repeating it for my control group as well. So I'm sitting with a baked potato or whatever idiom you want to use: I have to make the best of the results I have.
I'm using SYBR green and am picking up primer dimers.
So my target is low abundance at the start, but I could still get good enough results for the control group as primer dimer interference was minimal. However as soon as knock down occurred the primer dimers shot up and interfered with my Cq values. So I have decided to resort to the Tm peaks to analyze my results.
My reasoning is as follows: The Tm peak is drawn with a complicated equation that uses the amount of fluorescence in the sample as the temperature increases. When you compare samples with different starting concentrations of cDNA (C0), ie those in a standard curve, you see that their Tm peaks also differ in relation to each other - the sample with the highest C0 has the highest Tm peak and the one with the lowest C0 has the lowest Tm peak. This I can also see in my control sample versus my RNAi sample (see Tm pic1). Thus I should be able to get relative values for C0 by comparing their Tm peaks, right?
We're using a Roche Lightcycler 480 and it can give you the height as well as the area of a Tm peak. I've done this but am worried that it might not be calculating the areas correctly, especially for the small peaks. I am worried that instead of using area A (see Tm pic2) the program might use the sum of area A and B to calculate area.
Is there another (free) program that I can use to check these areas? Or do you have any other suggestions? Like the equation I might use with the peak height to calculate the area myself? Anything would be great and very appreciated!
I do realise that by using the Tm peaks it turns this whole qPCR analysis effectively into end-point analysis and thereby loses a lot of the accuracy of qPCR. But because my target is so low abundance, by the end of the qPCR cycles (I run 40 cycles) it is only just reaching the lag phase of amplification. (See Amp curves pic1)
I have emailed Roche support before and they agreed that I might be able to use the Tm peak area for this analysis if the samples are not well into lag phase - they gave the "okay" for the same pic I've given you. I will email them again now with this new problem and hear if they can shed any light on how their software calculates the peak, whether from height alone or some other way...
Thanks for all your help!
qPCR: Can I compare area under curve from Tm curves for quantification?
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