Problem in repeatability of Real time PCR results

Dear Friends,

We are working on HBV quantification by using Rotor gene with Artus HBV PCR kit, the problem here is we are trying to work in 1/4 of the recommended volume and it is not showing similar results with the full volume results.

We have taken 7.5 microliters of master mix and 5 microliter of Extracted DNA and the recommended volume is 30 microliter of master mix and 20 microliter of extracted DNA

The results shows as follows.

For 12.5 microliter volume reaction copies are 89180253 Iu/ml.

For 50 microliter reaction copies are 166873792 Iu/ml.

What our idea is if you add 4 volumes greated amout of DNA in the 50 microliter reaction we are expecting 12.5 reaction copies * 4..But as per our results it is showing only less thandouble amount of copies

Please let us know the problem and solution.

Phani.

Hi Phani,

Getting linear results when you use one fourth of the recommended volume of a qPCR reaction is not common. In other words using lower volumes works, but it will never give you the robust signal that a 50 ul reaction does. And to put it in yet another way, you cannot really compare the signals obtained in a 50 ul reaction that obtained from a 12.5 ul reaction, even if both came from the same master mix. 

I would recommend running a standard curve at both reaction volumes and determine if both reaction volumes give you the same slope and y-intercept. Most likely they will not. In that case, you should use the reaction volume that gives you the best results, which will also be the reaction volume with the most accurate quantification. 

Best

Dear Ivan,

Thank you.