I am trying to build up 3 standard curves for 3 sets of primers for different reference genes, in order to later use one with the best efficiency. (for SYBR green qPCR, gene copies number detection).
I use three reference genes ACT1, ARG4 and KanR - a single copy of each in my Pichia strain genome. I use 10-fold diluted series of template (10ng - 0.01 ng) .
The question is: am I supposed to see the same Ct for all primers , for each point dilution ? What I see now - a bit weird for me, because the efficiency is good enough for each pair of primers, however Ct's for each certain template dilution are different (2-3 cycles difference..)
How can this be explained?
Thanks for your comments!
A slight change in efficiency could be exaggerated over many cycles. Do you check specificity via melting curve? Is it indicating the formation of one specific product? You could increase your accuracy/specificity by using Taqman probes for copy number analysis.
If you’re efficiency is around 100%, and your melting curve is good, I don’t think you have reasons to be worried. However, using more references gene might make you feel more secure.
Best of luck
Thank you for our comments!
In reply to Simon, the melting curves look pretty good, maybe just a little non specific product formation with only highly diluted template (~0.01ng).
In reply to Sara, thank you, actually I intentionally designed the primers so that to keep the same length (+/- 10 bp). However, recently I brought up this question - whether the lenght of the products can affect the Ct, and there were the comments that it is not likely.
https://www.researchgate.net/post/qPCR_with_SYBR_green-should_the_amplicons_GOI_and_reference_be_the_same_length
Moreover, there was a paper cited, but I decided to be on the safe side and to go on with the same length.
http://www.pressesagro.be/base/text/v21n1/3.pdf
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