Icycler qpcr machine

Total RNA was extracted from cultured cells using an RNeasy Microkit, including DNase incubation, according to the instructions of the manufacturer (Qiagen). One microgram of total RNA was reverse transcribed to complementary DNA (cDNA) using 0.5 μg of oligo(dT)_15‐18 primers, 1.0 mM dNTPs, 1× Reaction Buffer, 20 units of RiboLock, and 200 units of RevertAid reverse transcriptase (all from ThermoFisher Scientific) in a total volume of 20 μl per reaction. The cDNA product was diluted 10‐fold in water and used at this concentration for qPCR. Qualitative PCR was performed using SsoAdvanced Universal SYBR Green qPCR Master Mix (Bio‐Rad), with primers at a final concentration of 500 nM from a 10 μM stock. Diluted cDNA (2.5 μl) was used per reaction, and all reactions were performed in triplicate in a total volume of 10 μl. Primer sequences are shown in Supplementary Table 1, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art.40659/abstract. A 2‐step qPCR cycle with a Bio‐Rad iCycler qPCR machine was used for target amplification, according to the instructions of the manufacturer for SSoAdvanced Universal SYBR Green Master Mix, and CFX Manager was used for analysis.

RNA from purified neutrophils (un-stimulated or stimulated with gut microbiota) or small intestinal tissue was extracted using Trizol reagent and an RNeasy mini plus kit (QIAGEN). After quantification, RNA was used for cDNA synthesis using the iScript cDNA synthesis kit (Invitrogen). Samples were analyzed on an iCycler qPCR machine (Bio-rad). Gene expression level was determined using the 2^−ΔΔCt method and normalized with the housekeeping gene, Gapdh. Primers sequences are listed in Table 1. Each sample was assayed in duplicate and the experiments were repeated at least twice.