Table 2.
Relative quantification of different lengths of GAPDH mRNA relative to different lengths of APC mRNA as the standard
| conventional relative quantification |
corrected relative quantification |
||||||||
|---|---|---|---|---|---|---|---|---|---|
| GAPDH (bp) |
GAPDH (bp) |
||||||||
| APC (bp) | 81 | 100 | 166 | 257 | 81 | 100 | 166 | 257 | |
| expt 1 | 63 | 4.9 | 4.6 | 5.6 | 4.6 | 4.9 | 4.7 | 6.1 | 5.3 |
| control RNA | 85 | 8.8 | 8.3 | 10.1 | 8.3 | 8.7 | 8.4 | 10.7 | 9.4 |
| 0.7 lesions / 1000 bases | 161 | 9.2 | 8.7 | 10.6 | 8.7 | 8.6 | 8.3 | 10.7 | 9.3 |
| 218 | 7.1 | 6.7 | 8.2 | 6.7 | 6.4 | 6.2 | 7.9 | 6.9 | |
| degraded RNA | 63 | 11.2 | 7.5 | 6.0 | 3.5 | 12.6 | 9.4 | 11.4 | 11.9 |
| 6.3 lesions / 1000 bases | 85 | 14.7 | 9.8 | 7.8 | 4.6 | 14.4 | 10.8 | 13.0 | 13.6 |
| 161 | 34.8 | 23.2 | 18.5 | 10.9 | 21.0 | 15.8 | 19.1 | 19.9 | |
| 218 | 28.9 | 19.2 | 15.3 | 9.0 | 12.2 | 9.1 | 11.0 | 11.5 | |
| expt 2 | 63 | 3.9 | 3.8 | 2.6 | 2.8 | 4.1 | 4.2 | 3.3 | 4.5 |
| control RNA | 85 | 7.8 | 7.6 | 5.1 | 5.7 | 7.7 | 7.8 | 6.3 | 8.6 |
| 2.4 lesions / 1000 bases | 161 | 9.2 | 9.0 | 6.1 | 6.7 | 7.6 | 7.8 | 6.2 | 8.5 |
| 218 | 6.8 | 6.7 | 4.5 | 5.0 | 4.9 | 5.0 | 4.0 | 5.5 | |
| degraded RNA | 63 | 5.7 | 4.3 | 2.3 | 1.1 | 6.5 | 5.7 | 5.0 | 4.7 |
| 7.6 lesions / 1000 bases | 85 | 10.8 | 8.2 | 4.4 | 2.0 | 10.4 | 9.1 | 8.1 | 7.5 |
| 161 | 20.3 | 15.4 | 8.3 | 3.9 | 11.1 | 9.7 | 8.6 | 8.0 | |
| 218 | 14.7 | 11.2 | 6.0 | 2.8 | 5.2 | 4.6 | 4.0 | 3.8 | |
| expt 3 | 63 | 8.4 | 6.8 | 7.8 | 6.8 | 8.5 | 7.2 | 8.9 | 8.5 |
| control RNA | 85 | 13.4 | 11.0 | 12.6 | 10.9 | 13.3 | 11.2 | 13.9 | 13.4 |
| 1.2 lesions / 1000 bases | 161 | 19.7 | 16.1 | 18.5 | 16.0 | 17.9 | 15.0 | 18.6 | 17.9 |
| 218 | 12.0 | 9.8 | 11.2 | 9.7 | 10.2 | 8.5 | 10.6 | 10.2 | |
| degraded RNA | 63 | 15.2 | 10.1 | 8.2 | 3.7 | 17.2 | 13.1 | 16.9 | 14.2 |
| 7.0 lesions / 1000 bases | 85 | 24.6 | 16.3 | 13.3 | 5.9 | 23.9 | 18.1 | 23.5 | 19.7 |
| 161 | 50.8 | 33.7 | 27.5 | 12.2 | 29.0 | 22.0 | 28.4 | 23.9 | |
| 218 | 42.3 | 28.0 | 22.9 | 10.2 | 16.2 | 12.3 | 15.9 | 13.4 | |
Relative quantification was performed either by the conventional method or after quantifying degradation and then applying Equation (7). The RNA was either control RNA or RNA degraded by heating at 91°C for 30 min. The results obtained by conventional relative quantification are influenced by the length of the test amplicon, the length of the standard amplicon and the presence of degradation. These effects disappear after correction for degradation and length. For degraded RNA, in each experiment correcting the results of conventional relative quantification resulted in a highly significant (P < 0.0005) decrease in their variance; for control RNA correction produced a decrease in variance in each case but the decreases were not significant.