Superscriptiii first strand synthesis mix

Total RNA was isolated using NucleoSpin RNA isolation kit (Machery&Nagel), transcribed using SuperScriptIII First-Strand Synthesis Mix (Life Technologies) and subjected to qRT-PCR using predesigned TaqMan® Gene Expression Assay probes (ThermoFisher) on an AbiPrism 7500 Fast Sequence Detector. mRNA expression was normalized to RPLPO and relative changes in the respective mRNA were quantified by the 2^−ddCt method.

Cells from biological triplicates were harvested in TRIzol Reagent (LifeTechnologies) in weekly intervals and RNA was extracted according to the manufacturer’s instructions. Traces of contaminating DNA were removed using the TURBO DNA-free kit (LifeTechnologies). Synthesis of complementary DNA (cDNA) was performed using the SuperScript III First Strand Synthesis Mix (LifeTechnologies). For the non-enzyme control, a reference pool of all samples was used. cDNAs were subjected to specific target amplification prior to qPCR analysis using a pool of standard predesigned TAQman assays to be used later (Applied Biosystems) with a pre-amplification mastermix (Roche Life Science). Preamplified cDNA was diluted 1:5 and processed together with the TAQman assays for analysis in 96.96 Dynamic Array integrated fluidic chips on the BioMark HD platform (Fluidigm) according to the manufacturer’s instruction. Data were analyzed using Real-Time PCR Analysis software (Fluidigm).