Ecodry premix protocol at a glance

Cells or spheroids were harvested after transfection and RNA was isolated using the RNeasy Mini Kit 50 and QIAshredder 50 (QIAGEN, Hamburg, Germany). The final RNA concentration was measured using a NanoDrop Fluorospectrometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). An amount of 2 μg of total RNA was reverse transcribed using RNA to cDNA EcoDry Premix Protocol-At-A-Glance (Clontech, 2 Saint-Germain-en-Laye, France), the resulting cDNA was amplified using TaqMan Gene Expression Master Mix (Applied Biosystems, Vienna, Austria). The PCR products were analyzed on the Chromo4 PCR System (Bio-Rad, Hercules, CA, USA). The following TaqMan probes were used: GAPDH (Hs99999905_m1), IKBKG (NEMO; Hs00415849_m1), MAP3K14 (NIK; Hs00177695_m1), RELA (Hs00153294_m1), RELB (Hs00232399_m1), CREL (Hs00968440_m1), NFKB1 (p100; Hs00765730_m1), NFKB2 (p105; Hs00174517_m1), and MMP1(Hs00899658_m1). qPCR was performed in triplicate for each cDNA template. Gene expression normalized to GAPDH expression (glyceralaldehyde 3-phosphate dehydrogenase) and was calculated using the ΔΔC_T method.

Cells were harvested after transfection, and RNA was isolated using the RNeasy Mini Kit 50 and QIAshredder 50 (QUIAGEN, Hamburg, Germany). The final RNA concentration was measured using a NanoDrop Fluorospectrometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). An amount of 2 µg of total RNA was reverse transcribed using RNA to cDNA EcoDry Premix Protocol-At-A-Glance (Clontech, 2 Saint-Germain-en-Laye, France). The resulting cDNA was amplified using TaqMan Gene Expression Master Mix (Applied Biosystems, Vienna, Austria). The PCR products were analysed on the Chromo4 PCR System (Bio-Rad, Hercules, CA, USA). The following TagMan probes were used: GAPDH (Hs99999905_m1) and MYLK (Hs00364926_ma). qPCR was performed in triplicate for each cDNA template. Gene expression was normalised to GAPDH expression (glyceraldehyde 3-phosphate dehydrogenase) and was calculated using the ∆∆C_T method.