Where is my spike? - (Aug/26/2006 )
Last I got an unexpected QPCR results. I wanted to evaluate the accuracy of QPCR on a certain DNA sample with unknown concentration. So I performed QPCR with:
-Spike (with higher concentration than the interested DNA) alone (2)
-and the DNA + Spike (3)
I had expected QPCR would show me that set 3 had the highest concentration, or better, the exact sum of set 1 and 2. The result of set 2 is good and showed me the known concentration, but the result of set 3 reported me it has the concentration of set 2. Moreover, gel electrophoresis shows me the Spike disappeared when it is added to the DNA sample. It looks as if the DNA has "eaten" the Spike
Is there anyone who has spike experience with this strange case? What I have to consider with when I perform QPCR with spike in my DNA samples. All ideas are welcome.
With many thanks
I tried something similar: as I had small amount of starting material, I thought I can mix it with DNA of known concentration, then I will have the sum of quantity of the standard and quantity of the sample. I tried with samples with already measured quantity and I was quite surprised to find that the mixture has lower quantity than the standard.
I tried to figure out what's wrong - I found it depends on the method of analysis - second derivative max. or fit points (Light Cycler), so check if it will work in your case too.
P.S. If the spike is with much higher concentration than the DNA, I think you won't be able to quantitate it right.
Thanks for your reply. How did you fix this problem then. I don't quite get what you mean. And why can the instrument setting affect the results, since there is only one kind of raw signal (whether a reaction is spiked or not), I mean the strengthness of SYBR green fluorescence?
I agree with you that if the concentration of the spike is much higher than your sample, it is not possible to estimate correctly the concentration of your samples. But it does confuse me that that spike material reported even a lower concentration than spike only or sample only. I really cannot get this....