Iscript reverse transcriptase and random hexamer primers

Tissue RNAs were isolated using Direct-zol RNA kits according to manufacturer’s protocol (Zymo Research, Irvine, CA). Tissues were homogenized using nuclease-free 1.6-mm stainless steel beads in a Precellys24 homogenizer (Bertin Corp., MD, USA). 0.25 μg RNA was converted into cDNA using iScript reverse transcriptase and random hexamer primers (Bio-Rad Laboratories, CA, USA). Gene expression was determined by qRT-PCR using SsoAdvanced Universal Probes Supermix (Bio-Rad Laboratories). The primers and probes used in this study were purchased from IDT (Integrated DNA Technologies, IA, USA) and are listed in Supporting Information Table S1. All the threshold cycle (Ct) numbers were normalized to the reference gene Ywhaz.

Murine tissues were preserved in RNAlater (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA was isolated using Trizol (Thermo Fisher Scientific) or Direct™-zol RNA kits (Zymo Research Corp, Irvine, CA, USA) according to the manufacturer’s protocol. Tissues were homogenized using nuclease-free 1.6 mm stainless steel beads in a Bullet Blender (Next Advance, Inc., Averill Park, NY, USA). RNA (0.25–1 μg) was converted to cDNA using iScript reverse transcriptase and random hexamer primers (Bio-Rad Laboratories, Hercules, CA USA) according to the manufacturer’s recommendations. PCR reactions were performed with SsoAdvanced Universal Probes Supermix on a Bio-Rad iCycler iQ5 or CFX-96 QPCR system (Bio-Rad Laboratories). All the threshold cycle (Ct) numbers were normalized to 18S rRNA, -actin or Ywhaz. The probes and primers for the human CAMP and RN18S1 genes were described previously (38 (link)). Primers 5’-gtacatggctggggtgtt-3’ 5’-ttctacaatgagctgcgt gt-3’ and probe dCal Gold 540 5’-aggtctcaaacatgatctgggtcatct-3’ BHQ-2 for β-actin were purchased (Thermo Fisher Scientific and Biosearch Technologies, Novato, CA, respectively). Primer-probe mixes for Cyp24A1, Cyp27B1, and Ywhaz were purchased (Integrated DNA Technologies, Coralville, IA, USA).