Hypoxia-Induced Changes in mRNA Expression

Tumor cells were seeded at 10⁴ cells/well in 48 well plates in DMEM with 10% FBS. The cells were cultivated at either 20% or 2% oxygen (5% CO₂) for 48 h and harvested by passive lysis. RNA was isolated using the PeqGold RNA kit (Peqlab, Erlangen, Germany) according to the manufactures instructions.
mRNA-Expression was quantified by qRT-PCR on an 7900HT Thermal cycler (Applied Biosystems, Darmstadt, Germany) using SYBR green mix (Fermentas, Darmstadt, Germany). Sequences of primers used in qRT-PCR are given in supplemental table 1. All primer pairs were designed to span at least one intron to avoid amplification of contaminating gDNA. Expression levels of lox-family members were normalized against expression of rsp2960 (link).