Table 2

Effect of standard curve selection on E6, E2 and HMBS gene copy number calculations.

E6

Correlation Coefficient (R2)

Primer Efficiency (E)

Number of molecules at threshold (Nt)

E6 gene copy numbers over a range of theoretical crossing points


10

15

20

25

30


Final calibration curve for E6

0.9963

95.01%

3.3806E+11

4.25E+08

1.51E+07

5.34E+05

1.89E+04

6.72E+02


Replicate 1

0.9988

87.79%

1.43162E+11

2.62E+08

1.12E+07

4.81E+05

2.06E+04

8.82E+02

Replicate 2

0.9909

101.86%

5.85431E+11

5.21E+08

1.56E+07

4.64E+05

1.39E+04

4.13E+02

Replicate 3

0.9982

96.56%

3.68007E+11

4.28E+08

1.46E+07

4.97E+05

1.69E+04

5.77E+02

Replicate 4

0.9973

93.84%

2.55641E+11

3.41E+08

1.25E+07

4.56E+05

1.67E+04

6.09E+02


Coefficient of variation of replicates

28.72%

14.58%

3.83%

16.27%

31.32%

Mean copy number of replicates

3.88E+08

1.35E+07

4.74E+05

1.70E+04

6.20E+02

E2

Correlation Coefficient (R2)

Primer Efficiency (E)

Number of molecules at threshold (Nt)

E2 gene copy numbers over a range of theoretical crossing points


10

15

20

25

30


Final calibration curve for E2

0.9945

90.86%

2.42762E+11

3.79E+08

1.49E+07

5.90E+05

2.33E+04

9.02E+02


Replicate 1

0.9887

88.55%

1.92376E+11

3.39E+08

1.42E+07

5.96E+05

2.50E+04

0.9887

Replicate 2

0.9945

93.10%

2.8539E+11

3.96E+08

1.48E+07

5.49E+05

2.05E+04

0.9945

Replicate 3

0.9973

92.70%

3.11657E+11

4.41E+08

1.66E+07

6.25E+05

2.35E+04

0.9973

Replicate 4

0.9976

89.07%

1.81623E+11

3.11E+08

1.29E+07

5.33E+05

2.21E+04

0.9976


Coefficient of variation of replicates

15.69%

10.60%

7.36%

8.57%

13.06%

Mean copy number of replicates

3.72E+08

1.46E+07

5.76E+05

2.28E+04

9.02E+02

HMBS

Correlation Coefficient (R2)

Primer Efficiency (E)

Number of molecules at threshold (Nt)

HMBS gene copy numbers over a range of theoretical crossing points


10

15

20

25

30


Final calibration curve for HMBS

0.9970

91.05%

2.28115E+11

3.52E+08

1.38E+07

5.44E+05

2.14E+04

8.40E+02


Replicate 1

0.9951

89.10%

1.333E+11

2.28E+08

9.43E+06

3.90E+05

1.61E+04

6.67E+02

Replicate 2

0.9985

95.39%

3.52583E+11

4.35E+08

1.53E+07

5.36E+05

1.88E+04

6.60E+02

Replicate 3

0.9990

90.43%

2.4809E+11

3.96E+08

1.58E+07

6.31E+05

2.52E+04

1.01E+03

Replicate 4

0.9953

89.26%

1.78485E+11

3.03E+08

1.25E+07

5.13E+05

2.11E+04

8.71E+02


Coefficient of variation of replicates

27.36%

22.14%

19.17%

18.93%

20.99%

Mean copy number of replicates

3.40E+08

1.32E+07

5.18E+05

2.03E+04

8.01E+02


In the main part of each section the rows represent the values for the final calibration curve and for the four replicate qPCR runs of the 7 point NA6 titration series, from which the final curve was generated. The right-hand five columns give the gene copy numbers determined from each curve at theoretical crossing points ranging from 10 to 30. In the bottom part of each section the numbers represent the coefficient of variation in mean copy number determined between the four replicate qPCR runs, together with the mean copy number determined from replicates 1 – 4 at each Cp.

Roberts et al. BMC Biotechnology 2008 8:57   doi:10.1186/1472-6750-8-57

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