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Table 1 Transgene copy number determination by different methods.

From: Statistical tools for transgene copy number estimation based on real-time PCR

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

  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.