Itaq sybr

RNA was extracted from fibroblasts and placenta using the AllPrep DNA/RNA Micro Kit (QIAGEN) per manufacturer's instructions. cDNA was synthesized using the iScript™ cDNA Synthesis kit (Bio-Rad) with a consistent amount of RNA (500 ng for placenta and 70 ng for fibroblasts), assessed by the Qubit RNA HS Assay kit (Invitrogen) for each sample. Primers were manufactured by Integrated DNA Technologies and the primer sequences are listed in Supplementary Table S1. qRT-PCR gene expression quantification was performed using iTAQ SYBR (Bio-Rad) on the Bio-Rad CFX96 Touch Real-Time PCR Detection System with the following condition changes: 58°C annealing/extension. Results of the qPCR were analyzed using the Δ ΔC_T method (56 (link)).

ChIP was performed on chromatin isolated from patient-derived fibroblasts using the MAGnify Chromatin Immunoprecipitation System (Invitrogen) as per manufacturer recommendation. Chromatin was sonicated using the Covaris ME220 with the following parameters: PIP 75, DF 5%, CPB 200, 6°C setpoint for 16 min total, then precipitated using anti-CTCF (ab70303; Abcam) and rabbit IgG. To quantify CTCF occupancy, qPCR was performed with primers listed in Supplementary Table S1 using iTAQ SYBR (Bio-Rad) on the Bio-Rad CFX96 Touch Real-Time PCR Detection System with the following conditions: 58°C annealing/extension. Results of the qPCR were analyzed using the ΔΔC_T method (56 (link)).

In order to determine the effect of CR and TC on C. difficile genes involved in toxin synthesis, total RNA was isolated from the early stationary phase (12 h) cultures [5 (link)]. The culture supernatant was harvested by centrifugation at 3000× g for 10 min at 4 °C. The bacterial pellet was resuspended in RNAwiz solution (Ambion, Austin, TX, USA), flash frozen in liquid nitrogen, and stored at −80 °C. Total RNA extraction was performed using the Ambion RiboPure Bacteria RNA kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions, followed by DNase I digestion using Turbo DNase I (Ambion). The RNA obtained after each DNase I digestion was purified further using the Qiagen RNeasy RNA column purification kit, according to the manufacturer’s instructions (Qiagen, Germantown, MD, USA). The cDNA was synthesized using the Bio-Rad iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). RT-qPCR analysis of the genes associated with toxin production was performed using published primers for Paloc genes [46 (link)] normalized against 16S rRNA gene expression. Twenty-five μL reactions were performed in triplicate using iTaq SYBR (Bio-Rad, Hercules, CA, USA). The relative fold change in gene expression was calculated using the 2^−ΔΔCt method [47 (link)].