Doubts on Real Time PCR

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kiry's picture
Doubts on Real Time PCR




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Hei everyone!
First of all, I apologize if my English is not so correct!

I have just begun working on absolute Real Time qPCR and I have a problem with DATA analysis; in particular, from the assay,  I obtained a standards curve with a good efficiency (slope= -3,3; R2=0.999) but a very low sensitivity (y-intercept = 17). I know that to consider an assay with a good sensitivity, y-intercept has to be between 33 and 37. If it is higher than 37 , it indicates that the amount of template has not been correctly determined (for example because of  the deterioration of the template by too many freeze-thaw cycles of the standard), but what about if the value is too less than 33 as in my case (17)? However, this is not a case, I run the same assay for other samples, obtaining similar results of sensitivity and efficiency. What does it mean?
 I will appreciate  any suggestions, thanks!      

Ivan Delgado
Ivan Delgado's picture
Hi kiry,

Hi kiry,

You are correct in saying that your assay has very good efficiency, but I think you are mistaken when you say that your assay is not sensitive. A Ct value of 17 means that you have a lot of DNA in your sample or that your assay is very sensitive. 

Most qPCR instruments use as default PCR cycles 3 through 15 to define the baseline, so using a Ct value of 17 is perfectly fine. In an ideal world you want to use Ct value in the 20s, but anything above 15 for the most part is ok. I would even argue that any value above 33 is actually questionable since you start getting to very limited DNA amounts (assuming a 40 cycle PCR). 

When it comes to sensitivity, the bottom line is = what is the lowest DNA concentration in your standard curve that your assay can detect? I assume you ran at least 5 different DNA dilutions to create your standard curve. The lowest dilution your assay was able to detect, typically around Ct values in the mid 30s, defines how sensitive your assay is. For example lets say that your lowest DNA dilution was 10 picograms of DNA and you got a Ct value of 35 for it. Then your assay's sensitivity is 10 picograms. 

I hope this helps.