Hello everyone!
I am using AB Stepone system with Qiagen QuantiNova PCR kit. After doing my first trial, my qPCR results were very strange, the plot are "hook"-shaped (attachment). Anyone know what happen? The machine I use is not calibrated (the last calibration was around 2012~2013), will that cause any problem like this?
Our plate centrifuge are out of order and I cannot centrifuge properly, but no visible droplets remains on the wall. There are still some bubbles on the top liquid surface, is it a possible cause for my results (I thought the bubbles will be removed upon heating)?
I am doing a quantification experiment with 5 standards (300,000 copies, 1:10 diluted to 30 copies). I am new to qPCR and really confused by the result, please help :(
Thank you for your help!!
My guess is that you're not getting any amplification at all. The level of fluorescence you're seeing in the later cycles is very similar to the noise you're getting in the first couple cycles, when it should be orders of magnitude different. There's a "break" in the lines between cycles 3 and 10 because that's the "baseline" region; i.e., where the amplification is so small there should be no detectable increase in fluorescence. Subtracting this baseline is the "delta" in deltaRn. It would probably be really helpful to look at the graph of actual fluorescence value (instead of deltaRn) vs cycle number, to see what kind of amplification you're getting.
Hi Alan, I used the same software in the past and I remember that you can select other graphs to see the number of copies amplified and the fluorescence trend (even ROX trend if you used it) to verify, first of all, if you have amplification or not. As Stephen suggested can you post the graph of fluorescence value vs cycle number, other graph for better analysed your curves, and amplification problems.
I do not have experience with "quantitative" real time, for the "hook" phenomenon, I read something in the chapter I send you, paragraph 7.4.5, pag 20 in pdf, anyway all the chapter is interesting.
Hi Alan,
Centrifugation of your plate is very important, although no drops are on the walls. These can make a difference in the results. If you are not sure about what template concentration to use, I would go first for a standard curve with serial dilutions for each of your primers in order to know which one is the proper concentration for each one. If too concentrated, inhibition because of saturation of the reactives can be produced.
Hope it helps.
Hi Alan,
from the graph u put, I strongly agree with Stephen it seems just noises for me and one of the problems u mentioned could cause that. I think it might be due to the machine as q-PCR is very sensitive technique you might need to calibrate it first.
Good luck
I have similar problem. i am getting broken, zig-zak plots, no Ct values in qPCR machine. However, when I check the amplified product, I get perfect band. I checked with two different SYBR but getting similar results. In some sample I get perfect melting curve in most others there are multiple peaks. See image attached
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