Real-Time PCR Analysis of Gene Expression

Total RNA was isolated (n = 5–10 randomly selected from each group, cells n = 8–10) using TRI reagent (Sigma-Aldrich, MO, USA). First-strand cDNA was synthesized using M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant Kit (Promega, WI, USA) [20 (link), 21 (link)]. Pre-optimized TaqMan^® probe/primers (Additional file 1: Table S1, Life Technologies, CA, USA) and SYBR^® Green premiers (Additional file 1: Table S2, Bio-Rad, CA, USA) [22 (link)] were used for the real-time PCR (Eppendorf Realplex², Hamburg, Germany). The genes of interest were normalized against the housekeeping gene 18s rRNA (Additional file 1: Table S1). The average value of the control was assigned as the calibrator, against which all other samples are expressed as a fold difference.