Hello everybody,
I've had some problems with PCR efficiency a while ago (you can read the details here: https://www.researchgate.net/post/I_repeatedly_get_very_poor_PCR_efficiency_using_different_temperatures_and_dilutions-are_there_inhibitors?_tpcectx=qa_overview_following&_trid=yKC0TeiVC8dJhHgnuPjbND50_).
Since then I checked and rechecked (myself using different samples or by asking my colleagues about their experience) my primers, annealing temperatures, my dilution calculations, my RNA extraction, DNAse treatment and RT protocols, RT-PCR master mix and PCR machine. Apparently all was OK except reverse transcription (also it looks like our DNAse has some unspecific RNAse activity and DNAse treatment reduces cDNA amount but not efficiency).
My colleagues have told me that I most likely used a bad aliquot of random hexamer primers - some hapless student had mistakenly diluted them too much, and other people who used this aliquot experienced bad RT-PCR results, but they told me about increased Cts, not decreased efficiency (dCt between samples were as expected).
So, I have a question. How it's possible that overdiluted hexamers can change PCR efficiency?? I can only predict that with less hexamers you will have less cDNA, hence increased Cts. But low efficiency means that not all DNA molecules amplify in each cycle, and I can't wrap my head around how decreased hexamers concentration may cause it.
Hi
Its very simple. We describe molecules behaviors by (1/√n rule) that definite departure, when n is number of studied molecules or atoms. If you lower n, that means your describes have a lower resolution and accuracy. You overdiluted hexamers, then its possible that some hexamers related to your desirable genes are missed, so efficiency decreases.
Well it depends on how much your Cts have increased. Every reaction has its limits. It is a very different reaction if I have let's say 10 million copies of a particular cDNA and a completely different one if I have 100 copies of the same cDNA.
Georgios, thank you!
I, too, thought at first that I had too little cDNA and this quantity is out of dynamic range of the calibration curve. But I had 50% efficiency for GAPDH too, and it's very highly expressed housekeeping gene. Cts for GAPDH were from 18 (dilution 1:9 of sample cDNA) to 28 (dilution 1:729), and calibration curve was good and straight, with R^2>0.99 (see the graph attached). I don't know how many copies there were, though, these are relative calibration curves, but usually you should get within dynamic range if you have Cts less than 30, and you would see that your curve isn't straight anymore when you dilute the sample too much.
I used 4 primer pairs in this experiment, and all the calibration curves were strikingly parallel and their slope implied 50% efficiency (but Cts for other targets were more than for GAPDH, and some of them were >30). I already checked my dilution calculations, there wasn't a mistake.
A stupid question really, just because I see that your curves besides the low efficiency look quite good. Are you sure you have set the correct dilution at the software?
Well, yes, I'm sure =) There was dilution factor of 3, the most concentrated sample was 1:9, and then 1:27, 1:81, 1:243, 1:729.
You see different figures on "Quantity" axis (81, 27, 9, 3, 1) because the software operates with decimal fractions, and it's much easier to postulate that your most diluted sample is 1 arbitrary unit and then multiply to 3 correspondingly, than to calculate decimal form of 1/729 and set the inaccurate rounded number with many decimal places. When you use relative curve, it's only ratios between different points that are crucial for accurate calculations. I could set here, for example, 810, 270, 90, 30 and 10 and still have the same slope.
The only explanation I can think of is the presence of PCR inhibitors in your reactions but it cannot be from the random hexamers since the amount of cDNA you use in each reaction is different, therefore you would expect the effect to dwindle as the standard curve progresses. Did you run the exact same curve using the same aliquots from everything apart from your template? (i.e. do you have a past sample that you trust so that you can compare it with the ones you are using now?)
Well, I already got 98% efficiency for GAPDH using the very same RNA, so the RNA is good. I changed several factors: I designed another primer pair for this gene and redid reverse translation (without DNAse treatment) with fresh aliquot of hexamers, and all was good. Maybe I have the primers to blame, or the DNAse (but I also used this same DNAse with other samples recently, and efficiencies were OK), I have to check yet. But it's very peculiar that 4 different primer pairs had the same slope.
it is quite strange. Maybe the batch of water or TE you used to make the dilutions of the template? Or the dilution of the primers?
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