For microfluidic qPCR, cells were defrosted on ice and preamplification PCR completed using the following thermocycling protocol: 50 °C for 15 minutes, 95 °C for 2 minutes, 18 cycles of 95 °C for 15 s and 60 °C for 4 minutes, hold at 4 °C. Excess primers were removed with 3.6 μl of Exonuclease I from E. Coli according to manufacturer protocol (New England BioLabs, M0293S): 37 °C for 30 minutes, 80 °C for 15 minutes, hold at 4 °C. This final preamplified sample was diluted 5-fold in TE Buffer (TEKnova, T0224). Five microliters of diluted sample were loaded into each sample inlet of a 96.96 Dynamic Array chip (Fluidigm Corporation, San Francisco, CA, USA) and 5 μl from an assay mix containing DNA assay loading reagent as well as forward and reverse primers (10 pmol/μl), was loaded into each detector inlet. The chip was then placed in the NanoFlexTM 4-IFC Controller (Fluidigm) for loading and mixing. After loading, the chip was processed in the BioMarkTM Real-Time PCR System (Fluidigm) using a cycling program of 10 min at 95 °C
