First strand cdna synthesis system

AML cell lines KG-1a and OCI-AML-3 were collected after 5 days of culture at the appropriate oxygen conditions and total RNA was extracted with the Absolutely RNA Mini Prep Kit (Agilent Technologies, Santa Clara, USA) following the manufacturer’s instructions. For RT-PCR the First-Strand cDNA Synthesis System (Life Technologies, Carlsbad, USA) with the supplied oligo-(dT)₂₀ primer was applied. Quantitative PCR amplifications were performed using Fast SYBR Green Master Mix (Applied Biosystems, Waltham, USA) on the 7900HT Fast Real Time PCR system from the same company. For IL-8 cDNA amplification the following primers were used: human IL-8 forward 5′-TAGCAAAATTGAGGCCAAGG-3′ and reverse 5′-AGCAGACTAGGGTTGCCAGA-3′ (Biomol GmbH, Hamburg Germany). ß-actin was used as housekeeping gene: human ß-actin forward 5′-CCGAGGACTTTGATTGCACA-3′ and reverse 5′-AGTGGGGTGGCTTTTAGGAT-3′. (MWG Biotech, Ebersberg Germany). PCR conditions were: 95 °C for 15 min followed by 40 cycles of 95 °C for 15 sec, 60 °C for 30 sec, and 72 °C for 30 sec. Fold of RNA induction relative to normoxic conditions was calculated as the mean 2e^ΔΔCt of n = 4 independent experiments.

RNA from MC3T3 and C2C12 cells was isolated using a RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer instructions. First‐Strand cDNA Synthesis System (Life Technologies, Carlsbad, CA) was used for cDNA synthesis. qPCR was carried out using 2x Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific, Waltham, MA) in Fast 96‐Well Reaction Plates (Applied Biosystems, Foster City, CA) using a StepOnePlus (Applied Biosystems, Foster City, CA). Relative mRNA levels were calculated using the ΔΔCt method using 18srRNA for normalization. The qPCR primers are listed in Table S1.