Hi all,
I have a strange qPCR result here and I hope someone could help me on how should I interpret it.
In this case I am using probe-based qPCR, amplifying 2 targets at the same time. 1 endogenous gene (IPO8) and 1 viral gene.
In the figure attached, I highlighted the viral gene. The amplification for the endogenous gene looks good. However, the amplification plot for the viral gene looks weird as it goes up but reaches somewhat of a plateau then goes down before increasing again near the end of the qPCR.
Have someone got a qPCR plot like this before? How would you interpret it? How and where would you put the threshold for detection level of the gene?
Thank you.
either you have contamination in your mixing or there is something wrong with run steps
I've seen this before, though I don't know whether I ever managed to find a complete answer. I don't think contamination is an issue.
The first thing I would do is to reset the baseline calculation. Your 'wierd' samples are coming up very early, and many instruments automatically calculate the baseline from the first 15 cycles. In your case, 10 or less would be right. Also, take a look at the raw data in order to see your uncorrected amplification curve. It may also turn out that increasing the baseline cycles would help if the curve you are seeing is background.
The drop-off at ca. 28 cycles coincides with the maximum in the IPO8 curve. This suggests that there may be some kind of spectral interference between the colour channels. Check that your spectral calibration is up to date and that the right dyes and quenchers are specified.
Lastly, there is a hint of viral signal right at the end of the run. I'd add an extra five cycles in order to find out if it's real amplification or just noise.
Hi there! I don't know if you solved this already, but just in case I write. I had recently this problem and came to this post to try to find a solution, I found it elsewhere in the end, so I am writing for all the future newcomers as well. The problem with this is that the machine is reading the ROX value on your endogenous control. If you didn't add ROX to your samples (like me), this makes the machine subtract the values of your endogenous to all the samples, resulting in no amplification. Go to "set up" and, on the "assign targets to wells" tag, select the endogenous and select "none" in the reference. Then go to "analysis" and click on the "analyze" button. Magically, all your amplification curves will suddenly appear!
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