Whenever I am trying to run a qPCR analysis for a particular gene the exponential algorithm is failing. I have tried two different set of primers. Although the amplification curve is coming nicely for both the endogenous control and the gene of interest. The melt curve for the ensogenous control is also very good.
Dear Bodhisattwa,
Have you performed a primer efficiency experiment before starting? The first to check and easiest to troubleshoot is the primer efficiency. It is not clear now for me how you think exponential increase failed although you see a nice amplification curve. I assume, from your words, that you get increasing product with every cycle, but it is not doubled at each cycle. Then you should make a cDNA conc. gradient PCR and find out the efficiency of your primer pair (which is usually assumed to be 2, but should be found out experimentally for each primer pair).
But, even if your primer efficiency is not 2, say it is 1.4, then your product should increase 1.4-fold at each cycle to give a nice amplification curve (yet with a smaller slope), which is also exponential mathematically.
It could also be your product size. Ideal product size for qPCR with e.g. SYBR green, is 100-200bp (Although you can find others reporting up to 500bp). With smaller and larger product sizes, the incorporated dye fluorescence loses the linear correlation, thereby you fail to see the exponential increase in your signal.
Here yyou can find a nice and compact guide for qPCR troubleshooting:
http://www.bio-rad.com/de-at/applications-technologies/real-time-pcr-troubleshooting
Good luck!
Dear Muammer,
Thank you for your reply. No, I have not done a primer efficiency experiment.
I don't think it is a problem with the product size, because the primer is designed to amplify only 113bp. Anyhow thank you again for the help.
If you would attach a plot with the amplification curves, we might better spot a possible problem.
It could be a software setting issue if you think the amplification curves look good. Agree with Jochen, more details and images are needed for diagnosis....
I am uploading the amplification plot... The blue curve is my sample and the other one is the endogenous control beta-actin.
Well, I can see the issue that the delta Rn for your ASS target starts at 1 in the earliest cycles and then never goes above 1 at the latest cycles--so there does not appear to be any evidence for amplification from these curves. But this still may be a software setting issue. Have you tried changing the baseline setting (background subtraction) to see if the curves change? Or there may be a reaction mix issue. Are you using the correct SYBR mix for the instrument? The Rn is calculated by normalizing the signal (SYBR) fluorescence to the reference fluor in the reaction--usually Rox--and different instruments require different amounts of Rox. If the instrument does not have the correct reference fluor in the SYBR mix, the calculations will be incorrect. You can try to eliminate this issue with the data that you already have if there is some way to reanalyze the data by eliminating the Rox normalization step. The manufacturer of your qPCR instrument can help you with this and perhaps have other trouble-shooting suggestions. The beta-actin curves look a bit odd as well--with more of an S-shape than pure exponential--so changing the software settings might also be useful there. Did you do a melt curve analysis with this experiment? What did that show?
As David said: you will need to adjust the background correction/subtraction. It doesn't seem to work well. Can you provide the deltaRn values without background correction?
Also in line with David: Are you sure that the correct product was umplified (not only checked by melting curves, also use old-fashioned gels!)
In contrast to David, I don't think that ROX normalization is the source of the problem here.
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