2.7. RNA Isolation from Fibroblast, cDNA Synthesis and Gene Expression Analysis

Alongside the previously described RTCA assay, four conventional flat-bottom 96-well plates were seeded by following the exact same cellular distribution as used for the E-plates. Cells were grown under the same culture conditions and were later challenged by using the exact same pattern as for the RTCA assay. At the selected test intervals, 10, 20, 30 and 40 h after challenge, the culture medium was substituted with RNAlater (Life Technologies, Carlsbad, CA, USA) to preserve the RNA. After 24 h at 4·°C, the plates were frozen at −80 °C for later use. Total RNA extraction, cDNA synthesis and gene-expression analysis were performed as described earlier.