Quantification of Inflammatory Mediators in Paw Tissue

Paws were quickly frozen in liquid nitrogen and ground with Trizol reagent using a chilled mortar and pestle for quality RNA isolation (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s instructions. cDNA was synthesized from 2 μg total RNA using RNA to cDNA EcoDry Premix kit (Clontech, Palo Alto, CA, USA). qPCR was performed with ABI PRISM 7500 real time PCR system (Invitrogen) using SYBR Green PCR master Mix (Invitrogen). Sequences and product lengths for each primer pair were as follows: IFT20 (forward: 5′-AGA AGC AGA GAA CGA GAA GAT G-3′; reverse: 5′-CAC AAA GCT TCA TAT TCA ACC CG-3′, 156 bp); IL-1β (forward: 5′-ACA GAT GAA GTG CTC CTT CCA-3′; reverse: 5′-GTC GGA GAT TCG TAG CTG GAT-3′, 73 bp);^{34 (link)} IL-6 (forward: 5′-ATG GAT GCT ACC AAA CTG GAT-3′; reverse: 5′-TGA AGG ACT CTG GCT TTG TCT-3′, 139 bp)^{35 (link)} and TGF-β (forward: 5′-TGA CGT CAC TGG AGT TGT ACG G-3′; reverse: 5′-GGT TCA TGT CAG GAT GGT GC-3′, 170 bp).^{36 (link)} All of the reactions were run in triplicate and normalized to the housekeeping gene GAPDH.