Quantifying Nrf2 and CDKN2A Gene Expression

The PAXgene blood RNA kit (Qiagen, Hilden, Germany) was used to perform total RNA extraction from whole blood samples according to the manufacturer’s recommendations. The RNA samples were reverse transcribed into cDNA using Transcriptor First Strand cDNA synthesis kit (Roche, Basel, Switzerland), and a 500 ng total RNA sample was used for the single strand cDNA synthesis. Gene transcript level was quantified using LightCycler® 480 System (Roche, Basel, Switzerland). TaqMan Gene Expression Assays specific to Nrf2 (Hs00975961_g1, Applied Biosystems, Carlsbad, CA) and CDKN2A (Hs00923894_m1, Applied Biosystems, Carlsbad, CA) were used to determine Nrf2 and CDKN2A expression levels. Relative expression levels of Nrf2 and CDKN2A were normalized against GAPDH (Hs02758991_g1, Applied Biosystems, Carlsbad, CA) [51 (link)] and HPRT (Universal Probe Library Human HPRT gene assay, Roche, Basel, Switzerland) [45 (link), 52 (link)], respectively. All assays were performed in triplicate, and the comparative threshold cycle (CT) method was used to quantify relative gene expression [53 (link)]. To ensure the rigor of our measurements, the intra-assay coefficient of variation for the gene expression levels of Nrf2 and CDKN2A was tested respectively using the TaqMan Gene Expression Assays which demonstrated good reproducibility (3.8% and 5.1% for Nrf2 and CDKN2A, respectively).