First strand cdna synthase kit

Total RNA was extracted from cells and tissues with TRIzol reagent (Invitrogen, Grand Island, NY) as per the manufacturer’s instructions. One-centimeter of distal colon was collected and the whole tissue was processed for RNA isolation. RNA samples (2 μg) were reverse-transcribed to cDNA using the First Strand cDNA Synthase Kit (MBI Fermentas, Hanover, MD) with random hexamer primer. qPCR was performed on a CFX96 Touch Real-Time Detection System (Bio-Rad, CA) in a 25 μl volume using SYBR green Supermix (Bio-Rad, Hercules, CA). Amplification conditions were: 95°C for 3 min, 50 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 20s. The fold-changes in mRNA expression for targeted genes were relative to the respective groups of vehicle-treated mice after normalization to 18 s rRNA. Tissue homogenates of colonic whole tissues were prepared with RIPA buffer (Cell signaling technology, Beverly, MA) and in situ cytokine production of TNFα, IL-6, and IL-17A in the homogenates was measured using LEGEND MAX™ Mouse ELISA Kit (BioLegend, San Diego, CA) following manufacturer's instructions.

Frozen liver sections stored in RNAlater were removed from solution with sterile forceps and submerged in 2 ml TRIzol (Life Technologies, Grand Island, NY). Tissue homogenization, phase separation, and RNA precipitation were performed according to manufacturer’s instructions. RNA quantity and purity were assessed using a Thermo Scientific 2000 Nanodrop Spectrophotometer (Thermo Scientific, Wilmington, DE); all samples had A260/A280 between 1.8 and 2.0. Total RNA (2 μg) was reverse-transcribed to cDNA using the First Strand cDNA Synthase Kit (Fermentas, Hanover, MD) with random hexamer primers.