I am using realtime PCR approach to measure mtDNA copy number with mitochondrial tRNA or rRNA and nuclear B2M primers. I isolated DNA from both the samples control and treated then I did real time, the CT value of mitochondrial rRNA and tRNA are lower compared to nuclear B2M. How to express DNA content relatively is a problem for me. Anyone can you suggest how to get that and here I am attaching my file.
We have been using a version of delta Ct to calculate relative copy number.
2^([Ct(A)*E(A)]-[Ct(B)*E(B)]
where Ct= cycle threshold, E= efficiency, and A and B are your genes of interest.
We first generate standard curves by analysing a 10-fold dilution series to determine the efficiency of both reactions (value out of 100%). Then we measure the Ct of each gene in triplicate, and take the mean Ct value of each. Then put the efficiency and mean Ct values in the equation above.
The equation basically measures the difference in Ct between the two genes, and corrects for the difference in efficiency in each reaction.
Using your data (and lets assume your efficiency is 100% for each reaction):
control = 2^[(30.53*1)-(19.60*1)]
= 2^(10.92) = 1947.39 rRNA per B2M
treated = 2^[(30.45*1)-(22.29*10)]
= 2^(8.16) = 286.19 rRNA per B2M
Does that make sense? You really do need to determine the reaction efficiency - you should not assume they are 100%.
Hope that helps,
Steve
We have been using a version of delta Ct to calculate relative copy number.
2^([Ct(A)*E(A)]-[Ct(B)*E(B)]
where Ct= cycle threshold, E= efficiency, and A and B are your genes of interest.
We first generate standard curves by analysing a 10-fold dilution series to determine the efficiency of both reactions (value out of 100%). Then we measure the Ct of each gene in triplicate, and take the mean Ct value of each. Then put the efficiency and mean Ct values in the equation above.
The equation basically measures the difference in Ct between the two genes, and corrects for the difference in efficiency in each reaction.
Using your data (and lets assume your efficiency is 100% for each reaction):
control = 2^[(30.53*1)-(19.60*1)]
= 2^(10.92) = 1947.39 rRNA per B2M
treated = 2^[(30.45*1)-(22.29*10)]
= 2^(8.16) = 286.19 rRNA per B2M
Does that make sense? You really do need to determine the reaction efficiency - you should not assume they are 100%.
Hope that helps,
Steve
Hi Stephen ,
Thank you very much . If you dont mind can you explain me how to know the real time pcr efficiency in this particular experiment. As I new to this I could not solve this.
Thank you
Hi Ram Babu Undi,
You can also find details on the delta delta Ct calculation in
http://blog.mcbryan.co.uk/2013/06/qpcr-normalisation.html
and particularly the next link will help you for the efficiency question
http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_042380.pdf
regards
George
Hi,
Do you have a reference for the mitochondrial copy number calculation? Thanks
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