Gene expression analysis of multidrug resistance markers

RNA isolation of cell lines was performed using the NucleoSpin^® RNA Plus kit (Machery-Nagel (Allentown, PA, USA); 740984). RNA was converted to cDNA using superscript II reverse transcriptase (Invitrogen (Waltham, MA, USA); 1080-044). Gene expression of the resultant cDNA template was assessed by quantitative reverse transcription PCR (CFX384 RT-PCR machine; BIORAD (Hercules, CA, USA)) using iQ SYBR SuperMix (BIORAD; 1708884). Primer sequences were utilised as follows: YB-1 forward (5′ AAG AAG GTC ATC GCA ACG AAG 3′) and YB-1 reverse (5′ CTC CTA CAC TGC GAA GGT ACT 3′); ABCB1 forward (5′ CCC ATC ATT GCA ATA GCA GG 3′) and ABCB1 reverse (5′ GTT CAA ACT TCT GCT CCT GA 3′); ABCC1 forward (5′ TTC TCG GAA ACC ATC CAC GA 3′) and ABCC1 reverse (5′ CCT GTG ATC CAC CAG AAG GT 3′); GAPDH forward (5′ ATG TTC GTC ATG GGT GTG AA 3′) and GAPDH reverse (5′ CTC TTC TGG GTG GCA GTG AT 3′). The housekeeping gene GAPDH was used as a control to normalize the data and the relative mRNA expression level was calculated using ΔC_q/ΔΔC_q methodology [14 (link)].