Duplex ddPCR analysis of placental and fetal genes

Duplex ddPCR analysis of Slc6a4 and Slc22a3 in the rat placenta and Mao-a, Tph1, and Ido2 in rat placenta and fetal organs was performed as described previously [33 (link)]. Using the duplex feature, we were able to absolutely quantify the gene expression of target and reference genes simultaneously. Briefly, the duplex reaction mixture consisted of 10 µL of ddPCR™ Supermix for Probes (Bio-Rad, Hercules, CA, USA), 1 µL of each of the predesigned probe assays (FAM and HEX) (listed in Supplementary Table S1), and 0.5 µL of cDNA (50 ng/µL), in a total volume of 20 µL. Droplets were generated using a QX200 Droplet Generator and subsequently amplified to end-point using a T100™ Thermal Cycler in the following conditions: Single cycle of 95 °C for 10 min, followed by 40 cycles of 94 °C for 30 s and 60 °C for 1 min and a single cycle of 98 °C for 10 min. Droplet counting was performed in a QX200™ Droplet Reader and the concentration of the target gene was calculated using the QuantaSoft™ Software. For final data evaluation, wells with droplet numbers of less than 13,000 were excluded. Results are reported as the number of transcripts/ng of transcribed RNA. The QX200™ Droplet Digital™ PCR System, T100™ Thermal Cycler, and all consumables and reagents were obtained from BioRad, Hercules, CA, USA (unless otherwise stated).