RT-PCR

Total RNA was extracted from rat secretory and maturation EO cells as described (Lacruz et al., 2012). Briefly, cell homogenates were treated using RNeasy^R Micro Kit (Qiagen, United States) according to the manufacturer’s specifications and converted to cDNA using iScript^TM cDNA Synthesis Kit (Bio-Rad, United States). RT-PCR amplifications were done using SsoAdvanced^TMUniversal SYBR Green Supermix (BioRad, United States) on a CFX Connect^TM Real-Time System (Bio-Rad, United States). Primer specificity was confirmed by analysis of a melting curve. All experiments were done in triplicates. Beta-actin was amplified to standardize the amount of sample RNA. Relative quantification of gene expression was achieved using the ΔCT method (Pfaffl;, 2001). Supplementary Table S1 lists all primer sequences used.