Quantitative PCR Protocol for Innexin Gene Expression

qPCR was performed as described in [12 (link)]. In brief, reactions were performed in triplicate with each reaction consisting of 5 µL of GoTaq^® qPCR Master Mix (Promega), 400 nM forward and reverse primers, 40 ng cDNA, and nuclease-free water (total volume = 10 µL). Primers for each innexin and our reference gene (ribosomal protein S7, RPS7) were used as in Calkins and Piermarini [12 (link)]; see Supplementary Table S1. The reactions were then subjected to the following thermocycling protocol using a C1000/CFX96 real time system (Bio-Rad Laboratories, Hercules, CA, USA): initial denaturation of 95 °C (3 min), followed by 39 cycles of 95 °C (10 s), and 58 °C (30 s), ending with a melt curve cycle. qPCR results were analyzed using the ΔCt method [18 (link),19 (link)] and expressed as relative gene expression.