qPCR efficiency >> 100%

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qPCR efficiency >> 100%

Postby mightyphil2000 » Jul 14 2011 3:38 pm

Hello
I am running a multiplex qPCR reaction using VIC and FAM probes for two targets. I ran a 2-fold dilution series, starting at 32ng/ul down to 0.25ng/ul. The slopes from my regression of ct over log DNA amount were ~-1.45 and ~-1.40 for the two amplicons, indicating PCR efficiencies >300%. I am puzzled because I am copying a previously published protocol which the authors claim gave efficiencies of 90%.

What does it mean when you get efficiencies >>100%? What might I be doing wrong?

Would be extremely grateful for any help/advice on this!

thanks!

Philip
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Re: qPCR efficiency >> 100%

Postby r.rosati » Jul 14 2011 9:10 pm

This might happen when you have PCR inhibitors in your template. They get diluted out at low copy numbers, artificially raising efficiency.
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Re: qPCR efficiency >> 100%

Postby mchlbrmn » Jul 15 2011 12:45 am

...or, at the other end, if you have extra non-specific stuff coming up in the dilute samples it can lower their Ct's. You might see that on a gel of the PCR reaction.
You can check the various parts of the curve and see if you get a normal efficiency for one end or the other, or the middle of the curve. If you're lucky you may find your data falls in a good range of the curve.
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Re: qPCR efficiency >> 100%

Postby mightyphil2000 » Jul 15 2011 10:30 am

Thanks for your great replies!

Ok, so if I change the DNA sample and TE buffer I'm using for the dilutions, this might get rid of the inhibitor problem? Or at least give me a different efficiency.

Also, I checked the efficiency excluding DNA samples at extreme ends of the series (32 and 0.25). It didn't make a difference. It doesn't seem to matter which samples I exclude or include. I still get roughly the same efficiencies.

Also, when I use the software program LinRegPCR to calculate efficiencies within each individual well, I get average efficiencies of ~84% and 94% for my two respective amplicons. Not sure which calculation i should believe. I suppose this suggests there is something wrong with my dilution series.

Thanks!

Philip
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Re: qPCR efficiency >> 100%

Postby JMG » Jul 15 2011 12:57 pm

Hello mightyphil,

You are merely adding too much template is all - so your standard curves are almost the inverse of what you want.

Never use anymore than 3 or 4 ng/uL in your qPCR reactions.

Actually 2 ng/uL is the most concentrated you should use of your template in a reaction.

Do your 1:2 serial dilutions from 2 ng/uL on and you will be golden...
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Re: qPCR efficiency >> 100%

Postby mightyphil2000 » Jul 15 2011 1:03 pm

Thanks JMG,

If I exclude wells with quantities >4ng (so limiting to 0.25, 0.5, 1, 2, 4 ng ) I still get the same slopes, so not sure if that's the problem...
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Re: qPCR efficiency >> 100%

Postby JMG » Jul 15 2011 1:40 pm

mightyphil2000 wrote:Also, when I use the software program LinRegPCR to calculate efficiencies within each individual well, I get average efficiencies of ~84% and 94% for my two respective amplicons. Not sure which calculation i should believe. I suppose this suggests there is something wrong with my dilution series.


I guess I don't see what is wrong with your 84% and 94% efficiencies here...

The accepted range for qPCR is generally 80 to 110%...

You get >>100% efficiency when you use all of your dilutions for calculation correct?

But then you get 84 and 94%, respectively after you eliminate the extremely concentrated points.

The difference between "90%" expected and 84% and 94% observed is negligible in qPCR,
given the different labs and different hands and oh-so-slightly different subtle nuances
in sample preps...

e.g. your lab's efficiency for target A is 94% and for target B is 84%, theirs is 90% for both targets ...
just report it that way. I assume you are using the exact same master mix and the exact same machine
and the exact same primer/probe supplier too?
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Re: qPCR efficiency >> 100%

Postby mchlbrmn » Jul 16 2011 1:07 am

I think Phil may mean by, "when I use the software program LinRegPCR to calculate efficiencies within each individual well, I get average efficiencies of ~84% and 94%", that the software calculates an individual efficiency for each well based on the amplification curve. (Is that right?). I always wondered why that method wasn't used more, my system doesn't include it. Intuitively I like it, but I haven't researched it.
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Re: qPCR efficiency >> 100%

Postby JMG » Jul 16 2011 12:32 pm

I see...

And - I too, think that the LinReg approach on each amplification curve (to calculate efficiency on
a sample-by-sample basis) is the ultimate way to go. From what I understand, deciding where
background starts for each curve is a confounding issue... but, the Sisti, Guescini Cyo method
should be able to get around that... (would be great for all machines to be capable of that -- and
I think they are moving that direction)...

So, to the point of efficiencies >>>100%...

I have seen that before with both a SYBR Green assay of several targets, and also pre-formulated
AB assays for several targets. The AB assays giving >>100% E was later found to be a machine programming
glitch/error, while I still have entirely no explanation for the SYBR Green assay that exhibited >>100%
efficiencies for several targets - although the operator at that time was a first-timer with the procedure
and may have not performed the serial dilutions correctly...

Perhaps if you try serial 1:4 or serial 1:5 dilutions you may get better slopes...

Or, you are dealing with arithmetic addition to your signal due to [a heck of a lot of] degraded and/or
truncated DNA in your samples...

Are you certain there are no other things being amplified by your primers/probes for each target?

Are your samples of the exact same variety as in the publication you got your qPCR approach from?
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Re: qPCR efficiency >> 100%

Postby mightyphil2000 » Jul 18 2011 6:19 am

Hey guys,
Thanks for your replies.

Yes, the LinReg approach calculates efficiencies within each well using the amplification curve. That approach gives me average efficiencies I want, albeit with variation between wells. BTW, there are 24 wells in total: 8 dilutions (32ng to 0.25ng) done in triplicate. So, using LinReg I get 24 efficiency calculations x2 (for the repeat PCRs and for the single copy gene PCRs). The other approach I've used involves regressing the Ct values of the dilution series over the log amount of DNA. The slope of the regression is used to calculate the efficiency using the formula (efficiency = 10^(-1/slope)-1*100). Using the latter approach I get one efficiency calculation for the repeat PCR and one for the single copy gene PCR, which is in the range of 300-400 %.

I am going get a new DNA sample and redo the experiment, using alternative dilutions as you suggest and I'll also run a few gels to see if there's non-specific amplification.

Thanks for your advice once again!

Philip
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Re: qPCR efficiency >> 100%

Postby mchlbrmn » Jul 18 2011 11:53 am

I think many people first optimize PCR primers with SYBR green before using the probes. Then they can see a melt curve at the end of the cycling. I suppose that running it out on a gel is even better in discriminating PCR products, although more time consuming.
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Re: qPCR efficiency >> 100%

Postby mightyphil2000 » Jul 19 2011 10:07 am

Hey,

Ok, so I figured out what I was doing wrong. It's kind of embarrassing :) . But basically, I was using the wrong base in the back transformation formula for calculating the PCR efficiency. So the formula I was using (10^-1/slope) requires the DNA amount to be log transformed to the base 10 but I was regressing Ct over DNA amount on the natural log scale. oops! :D

Thanks everyone!

Philip
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Re: qPCR efficiency >> 100%

Postby JMG » Jul 19 2011 11:13 am

Excellent - thank you for letting us know what it was~!
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