A 10-ml reaction mix is normally prepared for each panel. It contains 16 TaqMan Universal master mix (Applied Biosystems, Foster City, CA), 16 RNase P-VIC TaqMan assay, 16 TaqMan assay for the target gene (900 nM primers and 200 nM FAMlabeled probe), 16 sample loading reagent (Fluidigm, South San Francisco, CA) and DNA with about 1,100-1,300 copies of the RNase P gene. 4.59 ml of the 10-ml reaction mix was uniformly partitioned into the 765 reaction chambers of each panel and the digital array was thermocycled on the BioMark system. Thermocycling conditions included a 95uC, 10 minute hot start followed by 40 cycles of two-step PCR: 15 seconds at 95uC for denaturing and 1 minute at 60uC for annealing and extension. Molecules of the two genes were independently amplified. FAM and VIC signals of all chambers were recorded at the end of each PCR cycle. After the reaction was completed, Digital PCR Analysis software (Fluidigm, South San Francisco, CA) was used to process the data and count the numbers of both FAM-positive chambers (target gene) and VIC-positive chambers (RNase P) in each panel.