Quantitative RT-PCR Analysis of Oxidative Stress Genes

As previously reported6 (link), the total RNA was extracted using Trizol reagent (Invitrogen). Five hundred ng of DNA-free total RNA was used to perform the reverse transcription with the 2-step RT-PCR kit (Takara Bio Inc., Japan)6 (link). The transcribed cDNA was utilized for PCR amplification with specific primers summarized in previous publication6 (link). The primers for NAD(P)H:quinone oxidase-1 (NQO-1) were: Forward, 5′-CCATTCTGAAAGGCTGGTTTG-3′, and reverse: 5′-CTAGCTTTGATCTGGTTGTC-3′ 42 (link). The primers for γ-glutamyl-cysteine ligase catalytic subunit (GCLC) were: Forward 5′-TTACCGAGGCTACGTGTCAGAC-3′ and reverse 5′-TGTCGATGGTCAGGTCGATGTC-3′; The primers for γ-glutamyl-cysteine ligase modifying subunit (GCLM) were: Forward 5′-AATCAGCCCTGATTTGGTCAGG-3′ and reverse 5′-CCAGCTGTGCAACTCCAAGGAC-3′ 43 (link). PCR products were separated on 1.2% agarose gels and visualized with ethidium bromide (EB). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was tested as an internal control.