Supercript 3 reverse transcriptase

RNA was extracted from tissues and HUVEC using the RNeasy kit (Qiagen). Supercript III Reverse Transcriptase (Invitrogen) was used for first strand cDNA synthesis. Quantitative real-time PCR was carried out using PerfeCTa SYBR Green Fastmix (Quanta Biosciences) on a Bio-Rad CFX96 system. Oligonucleotides used for human and mouse are listed in Supplementary Tables 3, 4, respectively.

Total RNA was isolated using NucleospinRNA kit (Bioke, Leiden, The Netherland) according to the supplier's instructions. RT was performed on 1 μg RNA using Supercript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). After denaturation at 95°C for 2 min, PCR was performed for 40 cycles (1 min at 95°C, 45 s at 63°C, 45 s at 72°C) using SYBR Green (Roche). Transcript levels were compared relative to b-Actin housekeeping genes using the following primers: ACTB-Fwd CAGAAGGATTCCTATGTGGGCGA; ACTB-Rev TTCTCCATGTCGTCC
CAGTTGGT; EGFR-Fwd GTGATCCAAGCTGTCCCAAT; EGFR-Rev ACTGGTTGTGGCAGC
AGTC; ERBB2-Fwd TGTGTGGACCTGGATGACAA; ERBB2-Rev GATGAGGATCCCAAAG
ACCA; ERBB3-Fwd TGGGGAACCTTGAGATTGTG; ERBB3-Rev GAGGTTGGGCAATGGTAGAG