Colon RNA extraction and cDNA synthesis

Approximately 30–40 mg of colon tissue was used for total RNA extraction using TRIzol (Gibco BRL, Life Technologies, NY, USA), according to manufacturer’s instructions. Quality and quantity of RNA were determined by measuring the absorbance at 260 and 280 nm using NanoDrop ND-1000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). All samples had an absorption ratio A260/A280 greater than 1.8. RNA (1 μg) from each sample was treated with RQ1 RNase-Free DNase® (Promega Corporation, Madison, WI, USA), according to the manufacturer’s instructions, to remove genomic DNA contamination. Reverse transcription was performed using SuperScript VILO cDNA Synthesis Master Mix (Invitrogen, Grand Island, NY, USA), according to the manufacturer’s instructions, in an Eppendorf Thermo cycler at 25°C for 10 min, followed by 42°C for 60 min, and 85°C for 5 min. Samples were then cooled to 4°C. cDNA samples were stored at -20°C for RT-qPCR analysis.