
Set 1

Set 2

Set 3

Set 4


sigm/Δct/4par

65.5 (36.9)

106.4 (7.0)

108.3 (16.6)

84.9 (7.6)

sigm/Δct/5par

73.5 (21.9)

77.2 (4.5)

89.3 (4.6)

75.9 (8.3)

sigm/F0/4par

46.6 (35.8)

79.7 (15.2)

117.3 (79.1)

68.8 (21.1)

sigm/F0/5par

70.4 (42.4)

86.1 (17.8)

87.1 (33.6)

72.6 (48.6)

exp/Δct/4par

85.7 (21.8)

104.2 (11.2)

318.8 (31.7)

339.5 (60.7)

exp/Δct/5par

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.

wol/Δct/4par

59.4 (18.2)

83.9 (5.4)

105.7 (19.5)

74.1 (20.7)

wol/Δct/5par

57.5 (18.3)

81.9 (5.0)

103.9 (14.9)

73.9 (21.8)

wol/F0

53.5 (25.9)

76.3 (14.3)

310.2 (118.2)

295.8 (175.6)

calib/Δct/4par

126.0 (21.6)

100.6 (5.8)

100.6 (11.5)

99.9 (7.7)

calib/Δct/5par

128.6 (20.8)

97.9 (5.1)

99.3 (5.9)

99.3 (8.1)

 Five commonly used quantification methods in conjunction with threshold cycles estimated from four and fiveparameter 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 fiveparameter sigmoidal models (4par, 5par) were used in combination with the following methods: sigmoidal model with Δct method (sigm/Δct), exponential model with Δct method (exp/Δct), windowoflinearity method with Δct method (wol/Δct) and calibration curve with Δct method (calib/Δct). Methods for calculation of ratios based on the initial template fluorescence (F0) were: fourparametric sigmoidal model (sigm/F0/4par), fiveparameter sigmoidal model (sigm/F0/5par), exponential model (exp/F0) and the 'windowoflinearity' method (wol/F0).
 Numbers in bold are combinations in which the fiveparameter model performs best. N.V. : no realistic estimation values due to problematic fits.