Gene Expression Analysis
CCO cells were seeded on 100mm plates (Corning, Fisher Scientific) at ~2.5x106 cells per plate for 24 hours. Following a media exchange to L-15, 1% FBS for 24 hours, cells were treated with different amounts of cfIL-22 or pBK plant background control for varying time periods. Cells were collected by media removal, enzyme treatment (TrypLE; Thermo Scientific) and cell scraper (Fisher Scientific). Cells were resuspended in treatment media, centrifuged at 250xg, and supernatant decanted. Cell pellet was rinsed in PBS pH 7.5, collected by centrifugation and cell pellet subjected to quick freeze with liquid nitrogen and stored at -80°C.
Total RNA was extracted using Ambion Purelink RNA mini kit (Thermo Scientific) and cDNA synthesized using Superscript IV Reverse Transcriptase kit with oligo dT primer (Thermo Scientific). Quantitative PCR (qPCR) primers were designed using PerlPrimer open-access software (Marshall, 2004) and commercially synthesized Custom DNA Oligos (Thermo Fisher Scientific). qPCR was carried out using Ssofast Eva Green Supermix (Bio-Rad) and samples and reagents robotically dispensed (Ep-motion 5075, Eppendorf, Hauppauge, NY) to 384 well white plates (USA Scientific). Reaction volumes of 10µl, primer concentration of 400 nM and cDNA concentration of 10ng was used. All qPCR was carried out on Bio-Rad CFX 384. Primer efficiencies were established using a 4-fold serial dilution of cDNA sample with 80ng DNA set as an upper limit. qBase+ software (Biogazelle) was used for reference gene selection, M-value, and gene expression analysis.