I've been making qPCRs with SYBR Green and in some of my reactions the efficiency is under 90% (between 80%-89%)
Can i use the ΔCt method to analyse my results? If not, what should i do?
Short answer: yes but I wouldn't.
Long answer: the ddCt calculation assumes 2^ddct, which is 100% effiency (log 2 during logarithmic amplification phase). You can adjust this calculation to account for your reduced efficiency, but with efficiencies that low I would rather not rely on the data and do some more work to further optimise conditions and/or primer design to achieve a higher efficiency. This will make your publications stronger and your thesis and data in general more robust.
Agree with Florian and Daniels suggestions. In addition you can get some help from the below-mentioned discussion thread on the same topic:
https://www.researchgate.net/post/Low_efficiency_of_qPCR
best wishes,
SD
I agree with the above notes, nevertheless if you can correctly calculate the efficiency then the calculations are very simple to adjust. If - say - you have 85% efficiency then you should simply use 1.85^ddCT.
You can of course adjust your efficiency in downstream calculations just by using the formula Q=E^ddCT, where E = is the amplification efficiency . Efficiencies with 90 % are good to use, however, the highest the efficiency, the better. Sometimes is just problems with pipetting or you just need to optimize a bit your RT-PCR conditions. Good luck !
Hi, I agree with previous comments that the closer to 100% the better but wanted to add a consideration.
In my opinion, as has been said, 80%-90% is not horrible (just not perfect) and the problem here is if your target gene (cMYC for example) has a very different efficiency that your reference gene (beta actin, 18S rRNA or ...), let's say 80% vs 95%. Here you may have errors in quantification since the ddCT methods assumes both are the same (100%).
This is even more important if you are comparing the expression of different genes (cMYC vs Oct4, GHR vs FGFR, ... all normalized using the same reference gene, beta actin for example) in this case if cMYC efficiency is 95% (great) but Oct4 efficiency is 80% (not perfect but good to go), when you normalize to beta actin expression with the ddCT method you would be underestimating the gene with lower efficiency, cMYC in the example.
Hope this comment helps, good luck!!!
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