Method for determining the efficiency of nucleic acid amplifications

A method for the relative quantification of a target nucleic acid relative to a reference nucleic acid and standardized with a calibrator sample comprising the steps of:

• a) preparation of a common or two separate dilution series of target nucleic acid and reference nucleic acid;
• b) amplification of the various dilutions of target nucleic acid and reference nucleic acid under defined reaction conditions, the amplification of the nucleic acid being measured in real-time;
• c) setting defined signal threshold values for the target nucleic acid and reference nucleic acid;
• d) determining the cycle number Cp to which the signal threshold values defined for the target nucleic acid and reference nucleic acid are exceeded in each dilution;
• e) determining a continuously differentiable function of the Cp values determined in d) as a function of the logarithm of the amounts used of target nucleic acid and determining a continuously differentiable function of the determined Cp values as a function of the logarithm of the amounts used of reference nucleic acid;
• f) determination of the Cp values of the target nucleic acid and reference nucleic acid in the sample to be analysed as well as in a calibrator sample;
• g) assignment of the Cp values measured in step f) to particular function values of the functions determined in step e);
• h) calculating the quotients of the function values from g) of the target nucleic acid and reference nucleic acid for the sample to be analysed as well as for the calibrator sample;
• and
• i) determination of the ratio of the two quotients from h) as a measure for the original amount of target nucleic acid contained in the sample.

A method for the relative quantification of a target nucleic acid relative to a reference nucleic acid and standardized with a calibrator sample comprising the steps of:

• a) preparing a common or two separate dilution series of target nucleic acid and reference nucleic acid;
• b) amplification of the various dilutions of target nucleic acid and reference nucleic acid under defined reaction conditions, the amplification of the nucleic acid being measured in real-time;
• c) setting defined signal threshold values for the target nucleic acid and reference nucleic acid;
• d) determining the cycle numbers Cp at which signal threshold values defined for the target nucleic acid and reference nucleic acid are exceeded in each dilution;
• e) determining a continuously differentiable function of the logarithm of the amounts used of target nucleic acid as a function of the Cp values determined in d) and determining a continuously differentiable function of the logarithm of the amounts used of reference nucleic acid as a function of the determined Cp values;
• f) determining the Cp values of the target nucleic acid and reference nucleic acid in the sample to be analysed as well as in a calibrator sample;
• g) assignment of the Cp values measured in step f) to particular function values of the functions determined in step e);
• h) calculating the quotients of the function values from g) of the target nucleic acid and reference nucleic acid for the sample to be analysed as well as for the calibrator sample;
• and
• i) determination of the ratio of the two quotients from h) as a measure for the original amount of target nucleic acid contained in the sample.