Standard curve

/Standard curve

Standard curve

2017-12-05T14:16:39+00:00 December 5th, 2017|

Obtained by plotting Ct values against log-transformed concentrations of serial ten-fold (log) dilutions of the target nucleic acid. Standard curve is obtained for quantitative PCR and the range of concentrations included should cover the expected unknown concentrations range. It is used to find out the dynamic range of the target (and/or normalizer), to calculate the slope (therefore, efficiency), r and R2 coefficients, precision (standard deviation), sensitivity (y-intercept) and also to help with quantitation. Ideally, the slope of a standard curve should be -3.32, R2 > 0.99 and the y-intercept around 40 (Ct). For proper evaluation of PCR efficiency, a minimum of 3 points (ideally 5 – 7) in triplicates over 5 to 7 logs (1/10 dilutions) of template concentration is necessary. Otherwise, even when the efficiency is 100%, mathematical manipulation is influenced by standard deviations and efficiency calculation may result in a value between 70% and 170%. A poor standard curve is usually due to pipetting errors (including calibration issues) or the presence of inhibitors in the reaction. If not, the primers/probes may need to be redesigned. See the ABI Guide for Standard Curve Experiments and ABI Publication: Understanding Ct Value.

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