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.