Here I have met a problem:
I ran my qPCR with "SYBR green master mix" with actin primer to quantify my data relatively in use of "delta delta Ct." The data shown here is strange, because there are two kinds of amplification curve presented here(as figure):
(1) the curve increases at cycle 3 slightly and then enter the "normal" amplification curve
(2) the curve which is random and "messy" before entering the linear and exponential phase of qPCR amplification curve
This data brought me a problem that the Ct value is becoming more strange and the software interpret the Ct value of first curve automatically about 8, and the second one is about 33. So, it becomes more hard for me to analyse and calculate the relative expression of my targeted gene.
The first question I come up with is that how does this error take place? What factors may be the reason causing the curve become messy? And, how can I avoid at the next run of qPCR?
The second part of question is that:
Excluding of re-run it again, I wanna know how to avoid this kind of data by adjusting the "baseline" or "some others values" in the software. If better, is there any manual that I can follow step-by-step to adjust it?
Thanks!
Hi,
first some questions, from the image you provided i assume you use applied biosystem equipment and sofware ? And at what dilution do you use your cDNA, 1/20, 1/40 ?
The good curve is (2) ! The (1) shows an error where your system take into acount the fluorescence noise at the begining of the pcr.
I encountered the same problem and honestly I find that it happen nearly random. I tried modifying cDNA and primer concentrations but nothing seems to make it through the problem.
My guess would be that it's a machine problem. No matter the manipulator, the primers or the qPCR set up, at some point our system started to give us those kind of results.
Fortunately this problem can be fixed pretty easily in applied software by manually setting the baseline start. From the picture I would say fix it near 20 cycles and you will see that the curve (1) magically become like (2). In general, fix the baseline start before the exponential part of the curve.
And suddenly your qPCR experiment can be analyzed and you don't need to start over ^^.
I think you got a problem in RNA extraction (poor RNA) or something like high inhibitor.
besides your CT is too high for B-actin, so try new extraction with high quality (Run on a 2% GEL is a good option), and check ROX if its necessary for your device.
BEst,
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