The time needed for the annealing temperature (60uC) to reach the extension temperature (72uC) was 3 sec, and time needed from the extension temperature to the denaturation temperature (94uC) was 4 sec. The cooling time from the extension temperature to the annealing temperature was 60 sec without a cooling fan and 14 sec when using a cooling fan. Since the cooling-time difference was significant (46 sec) and corresponds to 27 min difference in the overall assay time over 35 thermocycles, we decided to use a cooling fan in our real-time microchip PCR system. The total PCR time for 35 cycles (a single cycle: 50 sec at denaturation, 60 sec at annealing, and 60 sec at extension), including a 15 min initial hot-start step at 95uC, was 2 hr. 78% (99 min) of the total PCR time was taken for steps in 35 cycles, 12% (15 min) for the initial hot-start step, and 10% (12 min) for heating and cooling between each step. The total PCR time can be reduced by shortening the heating and cooling time between each step by using a smaller reaction chamber, however the reduction in time would only be 6 min if the heating and cooling time is reduced by 50%