High-Throughput Microfluidic Real-Time PCR

The BioMark™ real-time PCR system (Fluidigm, South San Francisco, CA, USA) was used for high-throughput microfluidic real-time PCR amplification using the 96.96 dynamic arrays (Fluidigm, South San Francisco, CA, USA). These chips dispensed 96 PCR mixes and 96 samples into individual wells, after which on-chip microfluidics assembled PCR reactions in individual chambers prior to thermal cycling resulting in 9216 individual reactions. Real-time PCRs were performed using FAM- and black hole quencher (BHQ1)-labeled TaqMan probes with TaqMan Gene Expression Master Mix in accordance with manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). Thermal cycling conditions were as follows: 2 min at 50 °C, 10 min at 95 °C, followed by 40 cycles of 2-step amplification of 15 sec at 95 °C, and 1 min at 60 °C. Data were acquired on the BioMark™ real-time PCR system and analyzed using the Fluidigm real-time PCR Analysis software to obtain C_t values (see Michelet et al. 2014 for more details [10 (link)]). Primers and probes were evaluated for their specificity against cDNA reference samples. One negative water control was included per chip. To determine if factors present in the sample could inhibit the PCR, Escherichia coli strain EDL933 DNA was added to each sample as an internal inhibition control, using primers and probes specific for the E. coli eae gene [13 (link)].