Cells were either treated or not with native LDL or Mox-LDL at a final concentration of 100 μg/mL for 24 hours. Total RNA was extracted from cells using TRIzol total RNA isolation reagent (Roche Applied Science) and reverse-transcribed using Reverse Transcriptase Core Kit (Eurogentec) according to the manufacturer's instructions. qRT-PCR was performed in 96-well plate format using SYBR Green-based detection on a Step-One-Plus machine (Applied Biosystems) with each 20 μL reaction containing ~50 ng cDNA, 0.3 μM sense and antisense primers, and ABsolute qPCR SYBR Green ROX Mix (Thermo Scientific). The plate was sealed and cycled under the following conditions: 95°C for 15 min, 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. Each reaction was performed in triplicate and mRNA levels of RPL27 were used for normalization. The relative expression levels of mRNAs were calculated using the comparative ΔΔCt method. PCR primers used for the quantification of HO-1 and RPL27 were as follows:
HO-1 forward primer 5′-AAGACTGCGTTCCTGCTCAAC-3′;
HO-1 reverse primer 5′-AAAGCCCTACAGCAACTGTCG-3′;
RPL27 forward primer 5′-ATCGCCAAGAGATCAAAGATAA-3′;
RPL27 reverse primer 5′-TCTGAAGACATCCTTATGACG-3′.