Quantitative RT-PCR analysis

Total RNA was prepared from cultured cells using TRIzol® reagent (MRC, TR 118) and was treated with RNase. Some of the RNA (1 μg) was then subjected to reverse transcription using random-hexamer primers and a cDNA synthesis kit (TaKaRa, RR036A-1). The resulting cDNA was subjected to quantitative PCR analysis with SYBR® Green (ABI, 467659) and mouse-specific primer pairs (forward and reverse). The sequences of the primers for mouse cDNA were as follows: Keap1, 5′-GGCAGGACCAGTTGAACAGT-3′ and 5′-GGGTCACCTCACTCCAGGTA-3′; Hmox1, 5′-GAGCAGAACCAGCCTGAACTA-3′ and 5′-GGTACAAGGAAGCCATCACCA-3; Gsta1, 5′- TGCCCAATCATTTCAGTCAG-3′ and 5′-CCAGAGCCATTCTCAACTA-3′; Ulk1, 5′- TCGAGTTCTCCCGCAAGG-3′ and 5′-CGTCTGAGACTTGGCGAGGT-3′; Srxn1, 5′-GGAAGGAAGAAAGGAGATGG-3′ and 5′-AGAGTTCAGGCTATGGGGAT-3′; Nqo1, 5′-TTCTCTGGCCGATTCAGAG-3′, and 5′-GGCTGCTTGGAGCAAAATAG-3′; Fasn: 5′-GCTGCGGAAACTTCAGGAAAT-3ʹ; 5ʹ-AGAGACGTGTCACTCCTGGACTT-3ʹ; Srebf1: 5′-GGAGCCATGGATTGCACATT-3ʹ; 5ʹ-GGCCCGGGAAGTCACTGT-3ʹ; Rn18s: 5′-CGCTCCCAAGATCCAACTAC-3′ and 5ʹ-CTGAGAAACGGCTACCACATC-3ʹ. Rn18s ribosomal RNA was used as an internal control.