Quantitative Gene Expression Analysis

Quantitative Polymerase Chain Reaction (qPCR) was performed using TaqMan^® Probe-Based Gene Expression Assays supplied by Applied Biosystems, Life Technologies (Carlsbad, CA, USA). Individual TaqMan gene expression assays were selected for GADD45α, Survivin (Birc5: baculoviral inhibitor of apoptosis repeat-containing 5), and Bcl-2-associated X protein (BAX). Briefly, cells were lysed and RNA was isolated using a commercially available kit, QIAprep Spin Miniprep Kit, supplied by Qiagen (Valencia, CA, USA). Total RNA was quantified using a NanoDrop 2000 (ThermoFisher Scientific, Waltham, MA, USA). Reverse transcription of RNA to cDNA was then performed on a Veriti^® Thermal Cycler using the High Capacity RNA to cDNA kit supplied by Applied Biosystems, Life Technologies (Carlsbad, CA, USA). Quantitative real-time PCR was performed using 50 ng of cDNA per reaction well on a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Life Technologies, Carlsbad, CA, USA). Gene expression data was normalized to 18S ribosomal RNA housekeeping gene and calibrated to the control samples according to the 2^−ΔΔCt method as previously described [40 (link),49 (link),50 (link)].