Patent ID: 7930106

Claim:
A method of establishing an equation for an adjusted calibration curve, said method comprising the steps of: (a) obtaining a plurality of standard samples, each containing a constant starting quantity of an internal calibrator and a known starting quantity of an analyte polynucleotide standard; (b) coamplifying the internal calibrator and the analyte polynucleotide standard in each of the plurality of standard samples; (c) determining for each of the plurality of standard samples indicia of amplification for the internal calibrator and the analyte polynucleotide standard that coamplified, whereby there is obtained a collection of determined indicia of amplification for the internal calibrator as a function of the known starting quantity of the analyte polynucleotide standard, and a collection of determined indicia of amplification for the analyte polynucleotide standard as a function of the known starting quantity of the analyte polynucleotide standard; (d) optimizing a first equation to fit a first curve to the collection of determined indicia of amplification for the internal calibrator as a function of the known starting quantity of the analyte polynucleotide standard, thereby resulting in a first fitted equation; (e) optimizing a second equation to fit a second curve to the collection of determined indicia of amplification for the analyte polynucleotide standard as a function of the known starting quantity of the analyte polynucleotide standard, thereby resulting in a second fitted equation; (f) obtaining an adjustment calibrator comprising a predetermined quantity of the analyte polynucleotide standard and said constant starting quantity of the internal calibrator; (g) coamplifying the internal calibrator and the analyte polynucleotide standard of the adjustment calibrator; (h) determining for the adjustment calibrator indicia of amplification for the internal calibrator and the analyte polynucleotide standard that coamplified; (i) modifying the first fitted equation using the indicia of amplification determined for the internal calibrator of the adjustment calibrator, whereby there results an adjusted first fitted equation for fitted indicia of amplification for the internal calibrator as a function of the known starting quantity of the analyte polynucleotide standard; (j) modifying the second fitted equation using the indicia of amplification determined for the analyte polynucleotide of the adjustment calibrator, whereby there results an adjusted second fitted equation for fitted indicia of amplification for the analyte polynucleotide standard as a function of the known starting quantity of the analyte polynucleotide standard; and (k) establishing the equation for the adjusted calibration curve by mathematically relating to each other the adjusted first fitted equation, and the adjusted second fitted equation.