4 bromanisole

Tissue samples (jejunum, ileum and cecum) were cut into 20-mg pieces, immediately placed into RNA Later solution (Qiagen, Manchester, UK) and stored at −70 °C prior to RNA purification. A single tissue fragment was transferred into 1 mL of TRI Reagent (Molecular Research Center, Cincinnati, OH, USA), and homogenized using 2.7 mm of zirconium silica beads (BioSpec Products, Bartlesville, OK, USA) in a Magnalyser (Roche, Indianapolis, IN, USA). To separate the phases, 50 μL of 4-bromanisole (Molecular Research Center, USA) was added. The whole content of the tube was centrifuged and the upper aqueous phase was collected for total RNA purification using the RNAeasy mini kit (Qiagen, UK) following the manufacturer’s instructions. An Ambion^® Turbo DNA-free kit (Life Technologies, Carlsbad, CA, USA) was used for the treatment of RNA samples to remove genomic DNA. Both the purity and concentration of RNA were detected spectrophotometrically on a NanoDrop 200c (Thermo Scientific, Madison, WI, USA) and 1 μg of the total RNA was immediately reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). The resulting cDNA was 10× diluted in nuclease-free water (Qiagen, UK) and used as a template in a quantitative real-time PCR.