High-throughput qRT-PCR for Gene Expression

Total RNA was isolated using the RNeasyPlus Kit (Qiagen, Hilden, Germany) according to the manufacturer instruction. The quantity and purity of RNA was determined by measuring the optical density at 260 and 280 nm. 600 ng total RNA was reverse transcribed to cDNA with TaqMan Reverse Transcription Reagents (Applera GmbH, Darmstadt, Germany). For qRT-PCR the Fluidigm Biomark high throughput qPCR chip platform (Fluidigm Corporation, San Francisco, CA, USA) with pre-designed gene expression assays from Applied Biosystems was used according to the manufacturer instructions (Spurgeon et al., 2008 (link)). Data were analyzed using the ddCT method (Livak and Schmittgen, 2001 (link)) and expression values were normalized to the expression levels of the β-actin gene. All TaqMan assays are listed in the Supplementary Material in Data Sheets 1, 2 for stimulation with IL-1β/TNFα and BMDM supernatant, respectively.