LinRegPCR is a program for the analysis of quantitative RT-PCR (qPCR) data resulting from monitoring the PCR reaction with SYBR green or similar fluorescent dyes. The program determines a baseline fluorescence and does a baseline subtraction. Then a Window-of-Linearity is set and PCR efficiencies per sample are calculated. With the mean PCR efficiency per amplicon, the Cq value per sample and the fluorescence threshold set to determnine the Cq, the starting concentration per sample, expressed in arbitrary fluorescence units, is calculated. See this reference please: Ramakers et al., NeuroSci Lett 2003; Ruijter et al., Nucleic Acids Research 2009.

If you export your data (File→Save to Excel) you will get an excel file with all the data. In the output sheet you can see two colums with the N0(N0=Target quantity or starting concentration per sample, expressed in arbitrary fluorescence units), one of them calculated with the individual effiniency and the other with the mean efficiency for that plate.

thank all for your answers, i just try to find how can i transform this arbitary fluorescence units to a more understanding resutl like gene copies per liter.

If you need to know the exact number of copies, not just compare between groups, then you need to create a calibration courve, making reactions with known quantities of starting material. One possibility is:

1- make a PCR with primers more left and more right to your amplicon (amplicon X);

2- isolate and quantify this new amplicon (amplicon Y): first agarose electrophoresis and then isolation from the band in the gel (in column);

3- make a qPCR with different quantities of this starting material (amplicon Y at different dilutions);

4- calculate the quantity of your samples from this calibration curve.

But all this proccess (or maybe another better) only if you really need to know the exact amount of starting material in your samples, not just compare between groups.

thank you David

i have already done the quantification with the standard curve method because i need to know the quantities.

Then i tried to run my results with the LinReg. i have environmental samples and for dilutions i had use pure sample. So i want to see if with the LinReg could calculate quantities with the corrected efficiencies.

Hi Andreas,

It is not recommended to use the sample efficiency to correct the value of the sample amplification (I do not know why, reproducibility reasons, but there are references about that in LinRegPCR website). For example, conducting a qPCR to compare between groups, you have to use mean efficiency, not the efficiency of each sample.

I think you should plot N0 (calculated using mean efficiency) vs [DNA] (ug/uL OR number of copies/uL calculating the molecular mass of the sequence used for calibration curve), and then interpolate N0 values of the unknown samples (calculated using mean efficiency).

Regards