I agree with much of what Sachin has stated, but, you would preferably graph log of copies vs Cq values for the standard curve.  And, if you find that your target insert in the biological specimen extract and a purified template standard DNA for that same target/insert both amplify at the same (or very similar) efficiency, then you can use Xo = EAMP(b-Cq) to calculate the initial number of target copies per qPCReaction:

where Xo = initial number of target copies in the qPCReaction,

and EAMP = Exponential amplification value = EAMP = 10(-1/m)

(e.g if EAMP = 1.92, your target reaction amplification efficiency is 92%)

and b = y-intercept of the plot of log(copies) vs Cq

(e.g., b = the estimated/forecasted Cq value for 1 copy of target)

and Cq (quantification cycle) is the arithmetically averaged observed Cq value of the amplification of target in each your biological sample extracts from your transgenic plant unknowns.

However, should you find that your sample extracts amplify at a different efficiency than your purified standard does, you can use the attached files to reconcile the two slightly different universes.

If a different machine quantification threshold is used for the standard curve than is used for the biological extracts, the 2nd file attached below includes an adjustment to correct for that as well: 10(sample threshold - absolute standard threshold)

Hi abhishek,

Absolute quantification starts with correct concentration of your template DNA. Templated quantification with dye-based method is preferable. Take initially 2* 10^11 copies of your gene of interest (pcr product or plasmid) and dilute it to different concentration series from 10^10 to 10^3 copies (each must be taken into triplicate). Generate the standard curve which represents the straight line with ideal slope value of -3.32 (acceptable reange -3.50 to -3.1).  The graph you get will be concentration * ct values. The equation y=mx +c will be the general equation where m will be the slope and x will be copy number. Thus from this graph you can determine the copy number of unknown sample i.e. genomic DNA of transgenic plant.

I agree with much of what Sachin has stated, but, you would preferably graph log of copies vs Cq values for the standard curve.  And, if you find that your target insert in the biological specimen extract and a purified template standard DNA for that same target/insert both amplify at the same (or very similar) efficiency, then you can use Xo = EAMP(b-Cq) to calculate the initial number of target copies per qPCReaction:

where Xo = initial number of target copies in the qPCReaction,

and EAMP = Exponential amplification value = EAMP = 10(-1/m)

(e.g if EAMP = 1.92, your target reaction amplification efficiency is 92%)

and b = y-intercept of the plot of log(copies) vs Cq

(e.g., b = the estimated/forecasted Cq value for 1 copy of target)

and Cq (quantification cycle) is the arithmetically averaged observed Cq value of the amplification of target in each your biological sample extracts from your transgenic plant unknowns.

However, should you find that your sample extracts amplify at a different efficiency than your purified standard does, you can use the attached files to reconcile the two slightly different universes.

If a different machine quantification threshold is used for the standard curve than is used for the biological extracts, the 2nd file attached below includes an adjustment to correct for that as well: 10(sample threshold - absolute standard threshold)

Thank you so much Sachin and Jack for your helpful suggestions. 

Dear Jack,

I am trying to do absolute quantification for samples run on two different machines. I do have efficiency and Y-intercept from both machines, as well as an external standard curve done on one of the machines.

Would you be able to provide an explanation for the formula given in your second attachment? What do the variables stand for (Ts, Ta, Eamp, babs)?

Thanks a lot!

Ts = experimental sample derived standard curve dRn-scaled (y-axis) quantification cycle threshold value (number between 0 and 1),  Ta = absolute template derived dRn-scaled (y-axis) quantification cycle threshold value (number between 0 and 1).  Eamp = exponential amplification value (e.g. Eamp of 1.8 = 80% standard curve efficiency, etc.). ba = absolute curve y-intercept Cq value.  bs = copy transformed y-intercept Cq value for the experimental sample derived standard curve.

Xo = EAMP(b-Cq) = 10(Cq-b)/m)) 

In the relationship(s) above, when b (standard curve y-intercept) = the estimated Cq value for 1 initial reaction copy , then Xo = initial reaction copies...

Thank you, Jack!

 Thank you Jack M Gallup again, do you have any nice reference for that equation? 

Hi Lucie,

I have these equations in my 2011 book chapter:

“Difficult Templates and Inhibitors of PCR” in the book: “PCR Troubleshooting and Optimization: The Essential Guide” January 2011.

https://books.google.com/books?id=oXoUkTSbnFgC&pg=PA23&lpg=PA23&dq=Gallup+Kennedy+Oswald+qPCR&source=bl&ots=VWcqrekfEK&sig=sWAQn6HoSTIxSjxe059bhKzABgA&hl=en&sa=X&ei=1y6EUOd0gZzZBamKgKgG#v=onepage&q=Gallup%20Kennedy%20Oswald%20qPCR&f=false

These mathematical relationships are very easily derived from the equation of a line describing qPCR serial dilution amplifications when plotting log of dilution or log of known target copies versus Cq values... so they are highly non-mysterious equations.

Applied Biosystems User Bulletin #2  describes the "10((Cq-b)/m)" portion of the equation.  I merely used simple math to show that that relationship is also equal to the expression Rq or Xo = EAMP(b-Cq) is all.  However,  it took me several years to realize that it was all simple/straight-forward log-based math.  The stats involved in qPCR is the real Herculean task - something I am still very much struggling with.

How to calculate copy number from standard curve generated from various concentrations of pooled genomic DNA ?