Synergy 2 with gen 5

Liver samples from mice were flash frozen in liquid nitrogen and stored at −80°C prior to RNA extraction. Livers were homogenized in Lysing Matrix D tubes (MP Biomedicals) containing 700 μl RLT buffer with 1% (v/v) β-mercaptoethanol (QIAGEN) using a FastPrep-24 (MP Biomedicals) bead beater for 45 s at 6.5 m/s (repeated 3 times, incubating on ice for 1 minute in between rounds). Homogenates were centrifuged at 20,000 × g at 4°C for 5 minutes to pellet debris, RNA was extracted using phenol:chloroform:IAA, pH 6.7 (Sigma), and samples were mixed with 50% ethanol prior to being transferred to QIAGEN RNeasy columns, according to the manufacturer’s instructions. Cleaned RNA samples were subjected to DNase treatment (Invitrogen) prior to cDNA synthesis using an iScript cDNA synthesis kit (Bio-Rad). RNA was removed through the addition of 1 N NaOH at 65°C for 30 minutes, followed by the addition of an equal volume of 1 N HCl. cDNA was cleaned using a PCR clean-up kit (Promega) according to the manufacturer’s instructions. cDNA concentrations were adjusted to 1 ng/μl using the Synergy 2 with Gen 5 software (Bio-Tek) prior to qRT-PCR using iQ SYBR Green mix (Bio-Rad), gene-specific primers (Table S1), and a CFX96 qPCR cycler (Bio-Rad). Transcript abundance was calculated using the ΔΔCt method and data were normalized to β-actin gene expression.

RNA concentration was determined using the Synergy 2 with Gen 5 software (BioTek) and 2 μg was reverse transcribed by M-MLV reverse transcriptase (Fisher Scientific) in the presence of RNase inhibitor (Promega) and random hexamers (Promega). Reactions lacking the reverse transcriptase were used to control for DNA contamination. Newly created cDNA was diluted 1:100 and used in qRT-PCR using iQ SYBR green supermix (BIO-RAD). Amplification was achieved using a 3-step melt cure program on a CFX96 qPCR cycler (BIO-RAD). Transcript abundance was calculated using the ΔΔCT method normalized by the C. difficile rpoB gene and performed in biological triplicates. Each assay was repeated 3 independent times and a representative independent experiment is shown.