Method of quantifying polynucleotides using a stored calibration curve

A method for quantifying an analyte polynucleotide in a test sample by internal calibration adjustment, comprising the steps of:

• (a) obtaining at least coefficients of an equation for a first calibration curve prepared using a first apparatus that amplifies nucleic acids and monitors amplicon production as amplification is occurring to yield time-dependent indicia of amplification,
• wherein the equation for the first calibration curve comprises a baseline adjustment coefficient, and specifies the ratio of time-dependent indicia of amplification for an analyte polynucleotide standard to time-dependent indicia of amplification for an internal calibrator as a function of the starting quantity of the analyte polynucleotide standard input into a plurality of in vitro nucleic acid amplification reactions that coamplify the analyte polynucleotide standard and the internal calibrator, and
• wherein the plurality of in vitro nucleic acid amplification reactions are performed on at least the first apparatus, and each comprise a constant starting quantity of the internal calibrator and different known starting quantities of the analyte polynucleotide standard;
• (b) obtaining a first adjustment calibrator that comprises a first known quantity of the analyte polynucleotide standard and said constant starting quantity of the internal calibrator;
• (c) coamplifying the internal calibrator and the analyte polynucleotide standard of the first adjustment calibrator in an in vitro nucleic acid amplification reaction performed on a second apparatus that amplifies nucleic acids and monitors amplicon production as amplification is occurring,
• whereby there are determined time-dependent indicia of amplification for the internal calibrator and the analyte polynucleotide standard in the reaction comprising the first adjustment calibrator;
• (d) calculating the ratio of time-dependent indicia of amplification for the analyte polynucleotide standard to time-dependent indicia of amplification for the internal calibrator determined in step (c);
• (e) calculating a first adjustment factor as the difference between the ratio calculated in step (d), and
• a value calculated by solving the equation for the first calibration curve when the starting quantity of the analyte polynucleotide standard is set to the first known quantity of the analyte polynucleotide standard present in the first adjustment calibrator;
• (f) establishing an adjusted equation for a second calibration curve by modifying the baseline adjustment coefficient of the equation for the first calibration curve using the first adjustment factor calculated in step (e);
• (g) coamplifying the analyte polynucleotide of the test sample and said constant starting quantity of the internal calibrator in a test reaction performed on the second apparatus to determine time-dependent indicia of amplification for the analyte polynucleotide and the internal calibrator of the test reaction;
• (h) calculating the ratio of time-dependent indicia of amplification for the analyte polynucleotide to time-dependent indicia of amplification for the internal calibrator determined in the test reaction;
• and
• (i) comparing the ratio calculated in step (h) with the second calibration curve, thereby quantifying the analyte polynucleotide in the test sample.