1 µg of total RNA was reverse transcribed to cDNA in 20 µL reaction mixtures using an iScript Advanced cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) and a Bio-Rad T100TM Thermal Cycler (Hercules, CA, USA) according to the manufacturers’ instructions.
Absolute quantification of Slc22a3 transcripts in rat placenta (n ≥ 18) was performed by duplex droplet digital PCR (ddPCR) analysis, as previously described [32 (link)]. Briefly, each reaction mixture consisted of 10 µL of ddPCR™ Supermix for Probes, 1 µL of TaqMan®Slc22a3 probe (FAM; Rn00570264_m1, Thermo Fisher Scientific, Waltham, MA, USA), 1 µL of Ywhaz probe (HEX; qRnoCIP0050810, BioRad, Hercules, CA, USA) and 1 µL of cDNA (50 ng/µL), in a total volume of 20 µL. Droplets obtained using a QX200 Droplet Generator were amplified to end-point using a T100™ Thermal Cycler. Results, acquired using a QX200™ Droplet Reader, were evaluated using QuantaSoft™ software. For final data evaluation, only data obtained from wells in which the number of droplets obtained exceeded 13,000 were used. All instruments, consumables, and reagents used in this analysis were obtained from BioRad (Hercules, CA, USA), unless otherwise stated.
Absolute quantification of Slc22a3 transcripts in rat placenta (n ≥ 18) was performed by duplex droplet digital PCR (ddPCR) analysis, as previously described [32 (link)]. Briefly, each reaction mixture consisted of 10 µL of ddPCR™ Supermix for Probes, 1 µL of TaqMan®Slc22a3 probe (FAM; Rn00570264_m1, Thermo Fisher Scientific, Waltham, MA, USA), 1 µL of Ywhaz probe (HEX; qRnoCIP0050810, BioRad, Hercules, CA, USA) and 1 µL of cDNA (50 ng/µL), in a total volume of 20 µL. Droplets obtained using a QX200 Droplet Generator were amplified to end-point using a T100™ Thermal Cycler. Results, acquired using a QX200™ Droplet Reader, were evaluated using QuantaSoft™ software. For final data evaluation, only data obtained from wells in which the number of droplets obtained exceeded 13,000 were used. All instruments, consumables, and reagents used in this analysis were obtained from BioRad (Hercules, CA, USA), unless otherwise stated.
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