Qscript system

Wild-type (WT) and Nrf2-disrupted mouse embryonic fibroblasts (MEF) that were established according to previously published protocols [23 (link)] were seeded at 5 × 10⁵ cells/well in a 6-well plate. Cells were cultured in Iscove’s Modified Dulbecco Medium with 10% FBS. Cells were treated with TTCA (0.5 – 4 μM) or 10 μM SFN and were harvested 20 hours later. RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA) and quantified using UV spectrophotometry at 260 nm. An absorbance ratio of 260/280 was utilized to determine the purity of RNA. RNA integrity was confirmed by gel electrophoresis and cDNA was synthesized using 1 μg of RNA with the qScript system (Quanta Biosciences, Beverly, MA). Real time PCR was performed on a Bio-Rad My-IQ with SYBR Green (Bio-Rad, Hercules, CA). PCR efficiency was determined using a standard curve method and the Pfaffl method was used for quantification of fold changes [24 (link)]. Primer sequences for Nqo1 and Gapdh were as previously published [25 (link)]. SFN was obtained from LKT laboratories (St Paul, MN).

Total RNA was prepared from mouse livers using Trizol (Life Technologies) followed by DNAse digestion and application to the RNeasy kit (Qiagen, Valencia, CA). RNA quality and quantity was evaluated by electrophoresis and spectrophotometry at 260/280 nm prior to reverse transcriptase reaction. cDNA was synthesized with the qScript system (Quanta BioSciences, Gaithersburg, MD). Real-time PCR was performed on an iCycler-MyIQ (Biorad, Hercules, CA) using iQ SYBR green supermix (Biorad) in tetraplicate 20 μl reactions. The PCR efficiency was determined from standard curves and the Pfaffl method was used for calculations of fold changes [30 (link)]. Melt curves and agarose gel electrophoresis was utilized to ensure the specificity of the amplified product. Reactions were normalized to both glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and TATA binding protein (Tbp). Primer sequences were obtained from Primer Bank [31 (link)] and are shown in Table 2.