Whole transcriptome kit

We used qPCR (CFX96^TM, Bio-Rad) to analyze mRNA levels of target genes (normalized to Gapdh reference control) (Supplementary material, Table S3) and specific miRNAs (normalized to RNU6 reference control) (Supplementary material, Table S4), as previously described [10 (link),11 (link),14 (link)]. Briefly, total RNA was isolated (RNeasy mini kit; Qiagen^®, Germantown, MD, USA) from four specimens (two males and two females) of each experimental and control group, and its concentration and quality were determined by absorption at 260 nm, and ratios at 260/280 nm (>2.0), respectively (NanoDrop^TM 1000 spectrophotometer; Thermo Scientific).
To analyze mRNA levels, we performed reverse transcription to cDNA (Whole Transcriptome kit; Qiagen^®, Germantown, MD, USA), followed by qPCR analysis, using specific primers for mouse genome (QuantiTect^® primers assay, Qiagen^®, Germantown, MD, USA) (Supplementary Table S3).
To analyze miRNA levels, we used total RNA and miScript II RT kit (Qiagen, Louisville, KY) and performed reverse transcription synthesis, followed by qPCR analysis, using specific primers for mouse genome (miScript Primer Assays; Qiagen^®, Germantown, MD, USA) (Supplementary Table S4).
Relative mRNA or miRNA expression levels were estimated for each target gene or miRNA relative to the reference controls (Gapdh or RNU6) (ΔΔCt).

In order to quantify the mRNA expression levels of MMR genes under long term exposure of HM to TS carcinogens, we performed real-time qPCR analysis. After total RNA isolation, RNA quality and concentration were determined by absorption ratios at 260/280 nm (>2.0) and 260 nm, respectively (NanoDrop^TM 1000 spectrophotometer; Thermo Scientific). Subsequently, we performed reverse transcription to cDNA using a Whole Transcriptome kit (Qiagen^®, Louisville, KY, USA) and real-time qPCR analysis, using specific primers for target and reference genes for mouse (Msh2, Mlh1, Gapdh) or human (hMSH2, hMLH1, hGAPDH) (QuantiTect^® primers assay, Qiagen^®, Louisville, KY, USA) (Supplementary Table S2), and iQ^TM SYBR^® Green Supermix (Bio-Rad, Hercules, CA, USA), following the manufacturer’s instructions (each sample was assayed in triplicate). We used a Bio-Rad real-time thermal cycler CFX96^TM and PCR data was analyzed by CFX96^TM software (Bio-Rad, Hercules, CA, USA).