Mini opticon real time thermal cycler

Intestinal sections were homogenized in TRIzol (Invitrogen) and total RNA collected. RNA concentration was determined by Nanodrop and RNA integrity and DNA genomic contamination was assessed on a Bioanalyzer (Agilent). Only pure RNA samples with an RNA integrity number greater than 8.0 were used for further analysis. Total RNA was reverse transcribed into cDNA using random hexamers with Revert-aid MMuLV reverse transcriptase (Fermentas). 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. The target genes (CD55, Factor H, Factor B and C3, Table 1) Ct values were normalized to 18s, and the fold change compared to C57Bl/6 Sham was calculated as described previously (27 (link)). Melt-curve analysis of the PCR products ensured amplification of a single product.

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.