qPCR efficiency

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danushy
danushy's picture
qPCR efficiency

Hi, I am running a qPCR using absolute calibration to calculate copy number of my gene of interest. I have problems with the efficiency of the reaction. The r2 is great 0.999, so that part I assume is OK. But the efficiency is 70 or 80 %. I checked the primers, the annealing temperature is ok, in the melting curve I see only one pick, and the non template has a very small one, but when I run a gel, there is no product. So I am asking what can I do to improve my efficiency. Thanks.

Ivan
Ivan's picture
 

 
Hi danushy,
Low efficiency is typically a result of primer dimers or an assay that has not been properly optimized. It is also always possible that the assay was not designed properly. Do all the dilutions that make up your standard curve yield nice sigmoidal amplification curves? All the R2 tells you is that your pipetting technique is good so it says little about how well the assay is behaving. Could you provide more information about how you calculated your efficiency? What is the slope of your curve? I assume it is steeper than -3.8. Also, how did you set up your dilutions? Did you run 10-fold dilutions and prepared at least 5 different dilutions for your standard curve?
 

danushy
danushy's picture
Hi!!! Thank you for your

Hi!!! Thank you for your answer.
All the points of the standard curve have nice sigmoidal amplification curves, the slope is sometimes arround -4 and now is -3.874. So now it is better. But the efficiency is 81%.
As I told you, I checked the annealing temperature, twice.
The efficiency is calculated with the iCycler program when I do the PCR standard curve, I obtain the slope, the efficiency and the correlation coefficient. The only thing I observe is in the melting curve the non template has a very small pick arround 78 , andt the Tm of the product is 81.5. Could this be the problem? But when I run an agarose gel, there is no band!!! So I don′t undestand what is going on.
I do a 10-fold dilutions with 7 different dilutions. (100 pg, 50 pg, 10 pg, 1 pg, 0.1 pg, 0.01pg, 0.001pg)
Thank you for your help!!!!

Ivan
Ivan's picture
 

 
 
I think I have a better answer for your problem now. First of all, I strongly suggest you prepare a different set of dilutions (and I would not go lower than 10 pg; 1 pg if you must). I do not know what your DNA source is, but 0.001 pg is a very small amount of DNA. Considering that a single cell has 6 pg of DNA, and your target is a single copy gene, you cannot detect 0.001 pg of DNA. Along these lines, what Ct value do you obtain for your 0.001 pg dilution? If it is higher than Ct 35 then you are pushing your assay a little too much. 
I would not worry too much about the tiny peak in your negative control. As long as the signal is minimal, and the Ct of your negative control is significantly higher than the highest Ct on your standard curve, you should be fine. qPCR is a thousand fold more sensitive than an agarose gel so not being able to see a band in a gel does not really say much about how dirty your qPCR assay is. 
Another thing to consider, if you designed an assay against a sequence that has multiple copies in the genome you are studying, is that you may be amplifying more than one sequence at the same time (which may not be distinguishable in your melt curves). If this is the case, and your DNA template has a particular DNA sequence, you may end up with weird results like these. Unfortunately if that is the case, you may need to redesign your assay. This is unlikely, but possible.
 

mithu_du_bmb
mithu_du_bmb's picture
Would you please check your

Would you please check your product length of RT-PCR? Usually small DNA fragment is difficult to see in gel run.

RomeoLima
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Have you tried constructing

Have you tried constructing the standard curve just using  five points instead of seven? Selecting all the points can give you a good R^2, but at the same time if one of the points is reading beyond the actual limit of detection of your qPCR, then your efficiency can be as low as 70%. To make another set of standards is a really good idea!!!. I would suggest you to use instead of ten fold, a two or a five fold serial dilution.
 
The small peak that appear in your qPCR data could be due to primers dimers, since the melting temperature is lower than the corresponding to your target. If there is no Cq (or Cp) in your NTC, then do not worry to much  about that small peak!!

Good Luck
 
! - )

RL