|
Effect of standard curve selection on E6, E2 and HMBS gene copy number calculations. |
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| E6 |
Correlation Coefficient (R2) |
Primer Efficiency (E) |
Number of molecules at threshold (Nt) |
E6 gene copy numbers over a range of theoretical crossing points |
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|
|
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| 10 |
15 |
20 |
25 |
30 |
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|
|
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| 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 |
|
|
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| 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 |
|
|
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| Coefficient of variation of replicates |
28.72% |
14.58% |
3.83% |
16.27% |
31.32% |
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| Mean copy number of replicates |
3.88E+08 |
1.35E+07 |
4.74E+05 |
1.70E+04 |
6.20E+02 |
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| E2 |
Correlation Coefficient (R2) |
Primer Efficiency (E) |
Number of molecules at threshold (Nt) |
E2 gene copy numbers over a range of theoretical crossing points |
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|
|
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| 10 |
15 |
20 |
25 |
30 |
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|
|
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| 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 |
|
|
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| 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 |
|
|
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| Coefficient of variation of replicates |
15.69% |
10.60% |
7.36% |
8.57% |
13.06% |
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| Mean copy number of replicates |
3.72E+08 |
1.46E+07 |
5.76E+05 |
2.28E+04 |
9.02E+02 |
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| HMBS |
Correlation Coefficient (R2) |
Primer Efficiency (E) |
Number of molecules at threshold (Nt) |
HMBS gene copy numbers over a range of theoretical crossing points |
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|
|
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| 10 |
15 |
20 |
25 |
30 |
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|
|
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| 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 |
|
|
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| 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 |
|
|
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| Coefficient of variation of replicates |
27.36% |
22.14% |
19.17% |
18.93% |
20.99% |
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| Mean copy number of replicates |
3.40E+08 |
1.32E+07 |
5.18E+05 |
2.03E+04 |
8.01E+02 |
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|
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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