I'm performing rt-PCR on microRNA, in some reactions I have an efficiency >100 (200%) and in others it's < 100 (65%), I'm working with fast sybrgreen (applied biosystem), cDNA (1/100), primers [100nm], {95°C-30s/95°C-3s/60°C-30s), the melt curve is good, and the Ct (between 23-32), Can you please help to resolve this problem!
NB: I dont think that is due to inhibitors and the primers are optimized.
Hi Amine,
Are you sure that you always load same concentration of your template? since high or low concentration of template will have direct effect on PCR efficiency. i think it would be good if you use another housekeeping gene as a control beside your target gene. then compare the Ct and PCR efficiency changes between two genes in one sample.
Thank you for your answer, i'll try to change my housekeeping gene, and i'll compare.
In my own experience, I found out that DNA template dilution are not stable over time even at lower temperature. I use yeast total RNA to make my standard over 3-4 log.
How many log and what is the typical Ct values that you span?
In my experience, having an efficiency around 100% is great, but not critical. It sounds like your reaction is specific to your desired product and the crossing threshold values are in a good range. You can try to add an inhibitor (like DMSO) to your high efficiency reaction and/or Mg to your lower efficiency reaction to get them closer to 100%, but that may affect the specificity and acceptable range of your ct's.
Instead, I would use software that takes efficiency into account when calculating your relative quantities. That will allow you to use a technically sound reaction (that you already have) and give you accurate numbers to quantify your results. Even in the best circumstances (+/- 5 of 100%) it is important to calculate reaction efficiency and incorporate it into your analysis because over a number of cycles even that small deviation can have large effects on the numbers.
With microRNA I obtained efficiencies 81-89% (and I agree with BK that you should incorporate the correction into your analysis, some machines, like Viia7 provide this option on their software). I used a specific stem-loop primer for RT (as a first step) and then a real-time with Fw primer (derived from the stem-loop primer) a Rev primer (derived from the rest of the miR sequence) and a UPL probe. What are your primers like? I would assume that, if priming is dificult in begining - you could end up with the same effect as if having inhibitors (i.e. higher than 100% efficiency). All best.
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