ICC from KitcopGFP/+ mice were purified by FACS (Becton Dickinson FACSAria) using 488-nm excitation and a 530/30-nm bandpass filter for GFP, as previously described (61). Total RNA was isolated from ICC, and the mixed cell population was obtained after dispersion (pre-unsorted cells) from three mice (n = 3) using illustra RNAspin Mini RNA Isolation kit (GE Healthcare). Concentration and purity of RNA were measured using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). Comparative amounts of RNA were used for first-strand complementary DNA (cDNA) and synthesized using SuperScript III (Invitrogen), according to the manufacturer’s instructions. Polymerase chain reaction (PCR) was performed with specific primers using the AmpliTaq Gold PCR Master Mix (Applied Biosystems). The following numbers given in parentheses for the reference nucleotide sequence were used: Gapdh (NM_008084), Orai1 (NM_175423), Orai2 (NM_178751), Orai3 (NM_198424), Stim1 (NM_009287), and Stim2 (NM001081103). PCR products were analyzed on 2% agarose gels and visualized by ethidium bromide. Quantitative PCR (qPCR) was performed with the same primers used for PCR using SYBR Green Chemistry on the 7900HT Real-Time PCR System (Applied Biosystems). cDNA was prepared from three mice (n = 3), and each cDNA was tested independently with triplicate replicates. Negative (no template) controls were included in each qPCR run. Melting curve analysis was performed using the manufacturer’s standard protocol with no evidence for primer dimer or nonspecific products. Regression analysis using the serial 10-fold dilutions of cDNA was used to generate standard curves. Unknown amounts of mRNA were plotted relative to the standard curve for each set of primers and plotted graphically with Microsoft Excel. This gave transcriptional quantification of each gene relative to the endogenous Gapdh standard after log transformation of the corresponding raw data. In pilot studies, Gapdh was tested on ICC cells used in the present study and found to be an appropriate control for qPCR. Normalized values and SDs were calculated for differences of relative gene expression from four dilutions of technical duplicates from each animal.

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