Total RNA extraction and PCR analysis

Total RNA was isolated from cells using TRIzol reagent (Invitrogen, CA, USA) following the manufacturer’s recommendations, and subjected to DNase (Promega, Madison, WI, USA) treatment. Reverse transcriptase reactions were then performed by incubating 400 ng of total RNA with the First-strand cDNA synthesis kit (Takara, Dalian, China). Spectrophotometer analysis and agarose gel electrophoresis were used to assess total RNA concentration and purity. The cDNA was collected and amplified immediately by using the following primers: for Nrf2, 5'-TCAGCGACGGAAAGAGTATGA-3' and 5'-CCACTGGTTTCTGACTGGATGT-3'; for NQO1, 5'-ATGGTCGGCAGAAGAGC-3' and 5'-GGAAATGATGGGATTGAAGT-3'; for HO-1, 5'-TCTCCGATGGGTCCTTACACTC-3' and 5'-GGCATAAAGCCCTACAGCAACT-3'; and for GAPDH, 5'-AGATCCCTCCAAAATCAAGTGG-3' and 5'-GGCAGAGATGATGACCCTTTT-3'. The amplification and data acquisition were implemented on a quantitative real-time polymerase chain reaction (PCR) system (Agilent, USA) using FastStart universal SYBR green master (Roche, Mannheim, Germany). Conditions were preset at 95 ℃ for 10 min, followed by 40 circles at 95 ℃ for 15 s and 60 ℃ for 1 min. Relative quantification of mRNA expression was determined using the 2^−△△CT method. PCR amplification efficiency of each gene was established utilizing calibration curves. GAPDH was used as the internal reference gene. All the real-time PCR experiments were performed in accordance with the Minimum Information for publication of Quantitative Real-time PCR Experiments (20).