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Table 1 Summary for accuracy and precision of dilution ratio quantitation obtained from four independent dilution datasets.

From: Highly accurate sigmoidal fitting of real-time PCR data by introducing a parameter for asymmetry

  Set 1 Set 2 Set 3 Set 4
sigm/Δct/4-par 65.5 (36.9) 106.4 (7.0) 108.3 (16.6) 84.9 (7.6)
sigm/Δct/5-par 73.5 (21.9) 77.2 (4.5) 89.3 (4.6) 75.9 (8.3)
sigm/F0/4-par 46.6 (35.8) 79.7 (15.2) 117.3 (79.1) 68.8 (21.1)
sigm/F0/5-par 70.4 (42.4) 86.1 (17.8) 87.1 (33.6) 72.6 (48.6)
exp/Δct/4-par 85.7 (21.8) 104.2 (11.2) 318.8 (31.7) 339.5 (60.7)
exp/Δct/5-par 82.6 (21.2) 101.3 (10.2) 319.74 (29.9) 355.4 (61.3)
exp/F0 198.0 (93.1) 238.9 (92.6) 79.2 (134.0) N.V.
w-o-l/Δct/4-par 59.4 (18.2) 83.9 (5.4) 105.7 (19.5) 74.1 (20.7)
w-o-l/Δct/5-par 57.5 (18.3) 81.9 (5.0) 103.9 (14.9) 73.9 (21.8)
w-o-l/F0 53.5 (25.9) 76.3 (14.3) 310.2 (118.2) 295.8 (175.6)
calib/Δct/4-par 126.0 (21.6) 100.6 (5.8) 100.6 (11.5) 99.9 (7.7)
calib/Δct/5-par 128.6 (20.8) 97.9 (5.1) 99.3 (5.9) 99.3 (8.1)
  1. Five commonly used quantification methods in conjunction with threshold cycles estimated from four- and five-parameter sigmoidal models were applied for the analysis. Four different datasets differing in the number of replicates, enzymatic chemistry and platform were analyzed in respect to accuracy (average percentage of calculated ratios from real ratios) and precision (average c.v.; numbers in brackets). Threshold cycles estimated from the second derivatives maximum of four- and five-parameter sigmoidal models (4-par, 5-par) were used in combination with the following methods: sigmoidal model with Δct method (sigm/Δct), exponential model with Δct method (exp/Δct), window-of-linearity method with Δct method (w-o-l/Δct) and calibration curve with Δct method (calib/Δct). Methods for calculation of ratios based on the initial template fluorescence (F0) were: four-parametric sigmoidal model (sigm/F0/4-par), five-parameter sigmoidal model (sigm/F0/5-par), exponential model (exp/F0) and the 'window-of-linearity' method (w-o-l/F0).
  2. Numbers in bold are combinations in which the five-parameter model performs best. N.V. : no realistic estimation values due to problematic fits.