Rt2 first strand cdna synthesis

For in vitro analysis, PC3 cells were cultured in a 3D laminin-rich extracellular matrix (lrECM) containing 0.5 mg/ml Matrigel (lrECM; BD) and irradiated with a single radiation dose of 10 Gy or multifractionated radiation with either 5 fractions of 2 Gy (one fraction per day) or 10 fractions of 1 Gy (2 fractions per day) with a cumulative dose of 10 Gy. At 24 h after the final radiation dose, cells were collected and total RNA was extracted from samples of three different experiments using the miRNeasy Kit (Cat # 217004, Qiagen) according to the manufacturer's protocol and as published (4 (link)). For in vivo analysis, RNA was isolated using homogenized powder from snap frozen tumor samples kept at −80°C. Extracted RNA was subjected to complementary cDNA synthesis using RT2 first strand cDNA synthesis (Cat # 330404, Qiagen) according to the manufacturer's instructions. qRT-PCR was performed using RT2 SYBR Green qPCR Master Mix. The reaction (25μl) along with cDNA was aliquoted into the wells of RT² Profiler™ PCR Array Human PI3K-AKT Signaling Pathway (PAHS-058Z, Qiagen) which contains pre-dispensed, laboratory verified, specific primer pairs. Data were analyzed using the RT2 Profiler array analysis software version 3.5 (SABiosciences). Relative gene expression was calculated by normalization to the arithmetic mean of 5 housekeeping genes.