Perfecta sybr green fastmix reagent

One 2 cm jejunal section was snap froze in liquid nitrogen and stored at −80°C until used for RNA analysis. Total RNA was isolated using TRIzol reagent (Invitrogen) according to the instructions of the manufacturer. RNA quantity and quality was determined by using the Nano drop (Thermo Fisher Scientific). Total RNA (1 μg) was reverse transcribed into cDNA by using aScript first strand cDNA synthesis kit (Quanta Biosciences). Quantitative real-time PCR analysis was performed in 25 μl total volume using an Applied Biosciences StepOnePlus thermocyclers (Thermo Fisher) with Perfecta SYBR Green Fastmix reagent (Quanta Biosciences) using the following protocol: 3 min at 95°C; 50 cycles of 10 sec at 95°C, 20 sec at Tm (Table 1), 10 sec at 72°C; melt curve start at 60°C, increasing by 0.5°C up to 95°C. After amplification, relative gene expression was normalized to 18s rRNA values, and ΔΔCt fold change relative to sham-treated mice was determined.

Total RNA was isolated from the jejunum using TRIzol reagent (Invitrogen) according to manufacturer’s instructions. RNA integrity and genomic DNA contamination was assessed using a BioAnalyzer (Agilent) and quantity determined by Nanodrop evaluation. Only samples with no DNA contamination and RIN values greater than 7.0 were used for cDNA synthesis. Total RNA (1 ug) was reverse transcribed using qScript first strand cDNA synthesis kit (Quanta Biosciences) using random primers. Quantitative real-time PCR was performed in 25 ul volumes using a Mini-Opticon real time thermal cycler (Bio-Rad) and Perfecta SYBR Green Fastmix reagent (Quanta Biosciences) using the following protocol: 3m at 95°C; 50 cycles of 10s at 95°C, 20s at Tm (Table 1), 10s at 72°C; melt curve starting at 65°C, increasing 0.5°C every 5s up to 95°C. After amplification, Cox-2 and β2-GPI Ct values were normalized to 18s rRNA and then ΔΔCt fold change relative to Sham-treated wildtype mice was determined as described previously (30 (link)). Melt-curve analysis of the PCR products ensured amplification of a single product.

RNA collected from human samples was reverse transcribed using iScript reagent (Quanta Biosciences), RNA from cell lines was reverse transcribed using qScript (Quanta Biosciences). Real Time quantitative PCR was performed with the StepOnePlus Real-Time PCR System (Applied Biosystems) using PerfeCTa SYBR Green FastMix reagent (Quanta Biosciences). Primer sequences used are listed in Supp Table 4. For Western Blotting, cells were lysed in RIPA buffer (Boston BioProducts) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific), and Western Blot was performed according to standard procedures. Antibodies were purchased from Cell Signaling: c-Jun (60A8) Rabbit mAb; Bim (C34C5) Rabbit mAb; c-Fos (9F6) Rabbit mAb). Monoclonal anti-beta-ACTIN (AC-15) antibody was purchased from Sigma Aldrich. Fold change values were quantitated using ImageJ software and normalizing to ACTIN levels.