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The iCycler iQ Detection System for TaqMan Assays, Rev B

R baseline, the standard curve has a correlation coefficient (r2) of 0.995, but the 2 most concentrated samples showed little separation. This occurred because the default baseline cycles are cycles 210, and the most concentrated samples had already come above baseline before cycle 10 (see Figure 2). The baseline cycles should be limited to cycles in which none of the samples have come above baseline, so the baseline cycles were changed to include cycles 25 only. The quality of the standard curve could be further improved by changing the threshold to 900 RFU and including the last 75% of the data collected in each cycle. The final standard curve shows a correlation coefficient of 0.998 over a range of 8 orders of magnitude (Figure 3).

Sensitivity
The starting concentration of genomic DNA ranged from 100 ng to 781 pg in 2-fold dilutions, or approximately from 16,000 to 125 genomic equivalents, respectively, with 8 replicates for each dilution. These data were optimized by extending the baseline cycles to include cycles 221, setting the threshold to 30 and using the last 75% of the data collected during the annealing/extension step. The resulting standard curve (Figure 4) showed a correlation coefficient of 0.999, and the threshold cycles of each successive replicate group were separated from its predecessor and successor by at least 3 standard deviations, down to approximately 250 genomic equivalents (see table). Theoretically, the threshold cycles of each replicate group should be separated by exactly 1 cycle; in this experiment, the difference between threshold cycles ranged from 1.01 to 1.19 cycles with a mean separation of 1.07 cycles.


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