Pairwise efficiency: a new mathematical approach to qPCR data analysis increases the precision of the calibration curve assay

Table 3 Comparison of the accuracy between Pairwise Efficiency and the standard calibration curve method based on a chosen standard

Wells	Conc.	Efficiency	F0	Ratio (PE)	Error (%)	Ratio (Ct)	Error (%)
A1-A6	100 ng	0.73130	0.00800	1	N/A	1	N/A
A7-A12	100 ng	0.76200	0.00780
B1-B6	100 ng	0.77170	0.00660
B7-B12	100 ng	0.77230	0.00710
C1-C6	50 ng	0.83530	0.00280	2.513	20%	2.47	19%
C7-C12	50 ng	0.79550	0.00290
D1-D6	50 ng	0.81870	0.00290
D7-D12	50 ng	0.82390	0.00300
E1-E6	12 ng	0.75780	0.00060	8.519	6%	12.73	37%
E7-E12	12 ng	0.68420	0.00110
F1-F6	12 ng	0.72470	0.00090
F7-F12	12 ng	0.70420	0.00100
G1-G6	3 ng	0.76180	0.00020	35.455	10%	57.41	44%
G7-G12	3 ng	0.66870	0.00020
H1-H6	3 ng	0.72810	0.00020
H7-H12	3 ng	0.66640	0.00020	Aver error:	12%		33%

The efficiency of amplification of Actin beta was determined using Pairwise Efficiency or the standard calibration curve method (for standard method E values see Additional file 1: Table S3). The known dilution ratio (differences between DNA template concentrations) were used as a reference. 100 ng was taken as 1, and thus all diluted samples should have yielded the following values: 2 (for 50 ng), 8 (for 12 ng) and 32 (for 3 ng). The error values in determining the correct ratios were lower than those calculated by standard method. The average error for Pairwise Efficiency was equal to 12%, while the average error for standard method was equal to 33%

ISSN: 1471-2105