Quantitative Analysis of Cellular mRNA

Cellular nucleic acids from cultures in 6-well plates were extracted using the RNeasy Mini kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. qPCR analysis of cellular mRNA expression using Maxima Probe qPCR Master Mix including ROX dye (ThermoFisher Scientific, Dreieich, Germany), a StepOnePlus (Applied Biosystems, Foster City, CA, USA) quantitative PCR machine, and plasmid standard curves was performed as previously described [48 (link)]. Primers, Taqman probes, and cycling conditions are summarized in Table A1. Cellular nucleic acids from cultures in 96-well plates were extracted using NucleoSpin^® RNA Plus Kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s protocol. Relative expression levels of ISG54 and RPL13A were determined using QuantiTect SYBR Green RT-qPCR Kit (QIAGEN) with the respective specific primers on a LightCycler^® 480 Instrument (Roche, Basel, Switzerland). Relative mRNA expression levels were normalized to the housekeeping gene RPL13A and analyzed using the 2^(−∆∆CT) method, finally depicted as fold inductions over mock A, mock B, or medium, as indicated. Primers and cycling conditions are summarized in Table A2. Primer efficiencies have been tested before in 10-fold serial dilutions and were calculated to have >90% efficiency.