*4.9. RNA Isolation and Quantitative Real-Time (RT-qPCR)*

The total RNA from MDA-MB-231 and MCF-7 cells, respectively, was extracted using a Bioline ISOLATE RNA mini kit according to the manufacturer's protocol (Meridian Biosciences, London, UK). The quantity of the isolated RNA obtained was measured using a NanoDrop™ 1000 spectrophotometer (Thermo Fisher Scientific), and samples were stored at −80 ◦C. mRNA (600 ng) was reverse-transcribed to cDNA using a Precision NanoScript™ 2 Reverse Transcription kit (Primerdesign, Chandlers Ford, UK) according to the manufacturer's protocol. The samples were then placed in a thermocycler, which was set for 20 min at 42 ◦C, 10 min at 75 ◦C, and holding at 4 ◦C. The cDNA samples obtained were diluted 1 in 10 with RNase/DNase-free water and stored at −20 ◦C.

The cDNA samples were amplified in qPCR using the Thermo Scientific PiKoReal 96 Real-Time PCR system. For one reaction, a master mix containing 10 µL of PrecisionPlus™ 2 × qPCR Mastermix with Sybr green with inert blue dye (Primerdesign), 1 µL of the reverse primer, 1 µL of the forward primer, and 3 µL of RNase/DNase-free water was prepared. Five microliters of cDNA were first added to each well of a 96-well Piko PCR plate in triplicate, followed by 15 µL of the master mix, ensuring that no bubbles were formed in the wells. The expression levels were normalized to human β-actin and human GAPDH as the house keeping genes. RNase/DNase-free water was used as the negative control. The PCR primers were used as follow: β-actin forward nucleotide: 5 ′ -CTGGAACGGTGAAGGTGACA-3′ , reverse nucleotide: 5′ -AAGGGACTTCCTGTAACAATGCA-3′ ; GAPDH forward nucleotide: 5′ -TGCACCACCAACTGCTTAGC-3′ , reverse nucleotide: 5′ -GGCATG GACTGTGGTCATGAG-3′ ; ABCC1 forward nucleotide 5′ -CGACATGACCGAGGCTACATT-3′ , reverse nucleotide 5′ -AGCAGACGATCCACAGCAAAA-3′ ; and ABCC4 forward nucleotide 5 ′ -TGTGGCTTTGAACACAGCGTA-3′ , reverse nucleotide 5′ -CCAGCACACTGAACGTGATAA-3′ . The qPCR data were analyzed using the double-delta Ct analysis. Each reaction was performed three times.
