Faststart universal sybr green master rox master mix

Transcript levels were quantified using a two-step RT-PCR on the 7300 real-time PCR System (Applied Biosystems, Dublin, Ireland) with QuantiTect SYBR Green I (Qiagen) as a fluorescent dye. cDNA was reverse-transcribed from RNA with the high-capacity cDNA reverse transcription Kit (Applied Biosystems). Real-time PCR was performed with the FastStart Universal SYBR Green Master (ROX) master mix (Roche, Dublin, Ireland). The RT-PCR reaction conditions were as follows: 95 °C × 10 min, (95 °C × 10 s, 60 °C × 30 s) × 40, 95 °C × 15 s, 60 °C × 1 min, 95 °C × 15 s, 60 °C × 15 s. The primer sequences for the RT-PCR experiments were supplied by Sigma-Aldrich (Wicklow, Ireland) and were as follows: claudin-5 left, 5′--3′, and right, 5′--3′; β-actin left, 5′--3′ and right, 5′--3′. Relative gene expression levels were measured using the comparative C_T method (ΔΔC_T). Expression levels of target genes were normalized to the housekeeping gene β-actin.

RNA was isolated by Trizol extraction and cDNA was reverse transcribed from RNA (500 ng) with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Transcript levels were quantified on the StepOne Plus instrument (Applied Biosystems) with FastStart Universal SYBR Green Master (ROX) master mix (Roche). The RT-PCR reaction conditions were as follows: 95 °C × 2 min, (95 °C × 5 s, 60 °C × 30 s) ×40, 95 °C × 15 s, 60 °C × 1 min, 95 °C × 15 s, 60 °C × 15 s. The primer sequences for the RT-PCR experiments are supplied in Supplementary Table 2. Relative gene expression levels were measured using the comparative CT method (ΔΔCT). Expression levels of target genes were normalised to the housekeeping gene β-actin.