Table 1.

Transgene copy number determination by different methods.

Line	Zygosity	Method	LgRt(T/C)	Copy Num	Confidence Interval	Efficiency Adjust	Pred Copy	Exp Copy
1	Hemi-zygous	1	-1.05	0.49	(0.42, 0.56)	N/A	0.5	0.5
		2	-1.07	0.48	(0.33, 0.68)	No	0.5	0.5
		3	-0.93	0.53	(0.32, 0.42)	Yes (0.93)	0.5	0.5
		4	-1.00	0.5	(0.12, 2.23)	N/A	N/A	0.5
	Homo-zygous	1	-0.10	0.93	(0.79, 1.09)	N/A	1	1
		2	-0.06	0.96	(0.77, 1.19)	No	1	1
		3	0.01	1.01	(0.85, 1.21)	No	1	1
		4	-0.80	0.57	(0.24, 1.38)	N/A	N/A	1
2	Hemi-zygous	1	-1.10	0.47	(0.41, 0.52)	N/A	0.5	0.5
		2	-1.18	0.44	(0.21, 0.95)	No	0.5	0.5
		3	-1.25	0.42	(0.33, 0.52)	No	0.5	0.5
		4	0.06	1.04	(0.22, 4.99)	N/A	N/A	1
	Homo-zygous	1	0.19	1.14	(0.93, 1.40)	N/A	1	1
		2	-0.03	0.98	(0.81,1.19)	No	1	1
		3	0.18	1.13	(0.88, 1.47)	Yes (0.97)	1	1
		4	0.09	1.06	(0.31, 3.68)	N/A	N/A	1

The transgene copy numbers for both homozygous and hemizygous progenies of two transgenic lines were analyzed with real-time PCR and different statistical methods. Method 1 is the external standard curve design, Method 2 is the internal reference gene design with standard curve, Methods 3 and 4 are the internal reference gene design with fluorescence data. In Method 3, the cycle number was used as dependent variable, and in Method 4, the logarithm-transformed fluorescence signal was used as the dependent variable. Column 4 shows the logarithm-2 transformed ratio between the test line and control line, or between the transgene and internal reference gene. Column 4 presents the direct outputs from SAS code. Column 5 indicates the numeric estimation of copy number. The confidence levels for logarithm 2 transformed ratios are converted into confidence interval for transgene copy number and presented in Column 6. Column 7 indicates whether efficiency adjustment was included or not. The predicted and actual copy numbers were presented in Column 8 and 9.