Transcriptional Analysis of DNA Repair Genes

We used a real-time quantitative polymerase chain reaction (qPCR) analysis to evaluate the transcriptional levels of hMSH2 and hMLH1. Total RNA (RNeasy mini kit; Qiagen Inc., Valencia, CA, USA) was isolated from NCI and FaDu exposed to 1 μM or 2 μM of NNK and their corresponding untreated controls. Briefly, we determined RNA quality and concentration by absorption ratios at 260/280 nm (>2.0) and 260 nm, respectively (NanoDrop^TM 1000 spectrophotometer; Thermo Fisher Scientific, Waltham, MA, USA). We performed reverse transcription (iScript cDNA synthesis kit; Bio-Rad) from total RNA and real-time qPCR analysis (Bio-Rad real-time thermal cycler CFX96^TM; Bio-Rad) using specific primers for target genes and reference housekeeping gene, human glyceraldehyde 3-phosphate dehydrogenase (hGAPDH) (QuantiTect Primers Assays; Qiagen) (Supplementary Table S1), and iQ^TM SYBR Green Supermix (Bio-Rad). We performed assays on 96-well plates, in triplicate for each sample, and data were analyzed using the CFX96^TM software [59 (link),60 (link)]. Relative mRNA expression levels were estimated for each target gene compared to the reference control gene (ΔΔCt).