Critical evaluation of methods used to determine amplification efficiency refutes the exponential character of real-time PCR

Table 1 Comparison of amplification efficiency determinations generated by six different methodologies, based on four replicate standard curves

	Positional Analysis (Standard curve)		Fluorescence Analysis (Individual amplification profiles)
	*Serial Dilution (n = 6)*		*Sigmoidal*	*Exponential*
	*Linear Regression (E* _slope )		*Linear Regrn*	*Linear Regression*		*Nonlinear Regrn*
*[SG]*	C _t - E _slope	*LRE C* _1/2 - E _slope	*LRE E* _max *(n = 6)*	E _loglin *(n = 6)*	*"LinReg" (n = 6)*	*"Miner" (n = 6)*
0.5X	98.8% (0.9993)	97.9% (0.9999)	96.0% ± 1.7%	81.1% ± 1.6%	87.5 ± 3.5%	93.0 ± 1.8%
1.0X	96.2% (0.9994)	95.7% (0.9990)	95.6% ± 1.3%	79.8% ± 1.6%	89.6 ± 3.0%	90.6 ± 2.1%
1.5X	94.1% (0.9990)	93.5% (0.9995)	92.8% ± 0.9%	83.1% ± 1.4%	84.6 ± 4.2%	90.5 ± 2.8%
2.0X	90.9% (0.9989)	89.7% (0.9991)	85.6% ± 0.7%	79.2% ± 2.1%	90.8 ± 3.8%	87.2 ± 1.7%

Amplification efficiency was determined using four replicate standard curves supplemented with increasing quantities of SYBR Green I. The data is categorized based on the type of analysis. The bracket numbers under positional analysis are the linear correlation coefficients (r²) generated from each standard curve. The fluorescence analysis lists the respective average amplification efficiency ± standard deviation for the six target quantities used in the standard curve construction. [SG]:quantity of supplementary SYBR Green I, n: the number of profiles used in the construction of the standard curves.

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