pmol each primer/μl, primer sequences for rat and mouse are provided in Supplemental Data Tables 1–2). Pre-amplification was performed using a 10 min 95 °C denaturation step and 14 cycles of 15 s at 95 °C and 4 min at 60 °C. Reaction products were diluted 5 times in TE Buffer (Teknova, Hollister, CA, USA). Five microliters from a sample mix containing pre-amplified cDNA and amplification Master mix (20 mm Mgcl2, 10 mm dNTPs, FastStart Taq polymerase, DNA binding Dye loading reagent, 50× ROX, 20× Evagreen) was 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 BioMark™ Real-Time PCR System (Fluidigm) using a cycling program of 10 min at 95 °C followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s and 72 °C for 30 s. After completion of qPCR, a melting curve of amplified
