Quantitect primers assay

Quantitative real‐time polymerase chain reaction (qPCR) analysis (Bio‐Rad real‐time thermal cycler CFX96™; Bio‐Rad) was performed, as previously described⁷, ¹³, ¹⁶ and in Supplementary Material, to evaluate the effect of STAT3 knockdown or pharmacologic inhibition on transcriptional levels of EGFR, TNF‐α, IL6, STAT3, RELA(p65), REL, BCL2 and WNT5A, previously associated with HNSCC³², ³³, ³⁴, ³⁵, ³⁶, ³⁷, ³⁸, ³⁹, ⁴⁰ and in particular with bile carcinogenesis², ³, ⁴, ⁵ and HSCC.⁴¹ Specific primers were used for target genes and reference housekeeping gene, human glyceraldehyde 3‐phosphate dehydrogenase (hGAPDH) (QuantiTect Primers Assays; Qiagen; Supplementary Materials; Table S3) and data were analysed by CFX96™ software.⁷, ¹³, ¹⁶ Relative mRNA expression levels were estimated for each target gene compared to the reference control gene (ΔΔCt).

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).

We isolated total RNA (RNeasy mini kit; Qiagen Inc., CA, USA) from HHK and HHPC exposed to GDF with or without BAY 11-7082, and corresponding controls, to evaluate the transcriptional levels of RELA (p65), c-REL, bcl-2, TNF-α, ΔNp63, EGFR, STAT3, WNT5A, IL-1β and IL-6, using quantitative real time polymerase chain reaction (qPCR) analysis, as previously described [21 (link)]. Briefly, we determined RNA quality and concentration by absorption ratios at 260/280 nm (> 2.0) and 260 nm, respectively (NanoDrop™ 1000 spectrophotometer; Thermo Fisher Scientific, Waltham, MA). We performed reverse transcription (iScript cDNA synthesis kit; Bio-Rad) and real time qPCR analysis (Bio-Rad real time thermal cycler CFX96TM; Bio-Rad) using specific primers for target genes and reference housekeeping gene, human glyceraldehyde 3-phosphate dehydrogenase (hGAPDH) (Supplementary Table 1; see Supplementary information online), (QuantiTect Primers Assays; Qiagen), and iQ™ SYBR Green Supermix (Bio-Rad). We performed assays in 96-well plates, in triplicate for each sample, and data were analyzed by CFX96™ software. Relative mRNA expression levels were estimated for each target gene relative to reference gene (ΔΔCt). (Data were obtained from three independent experiments)

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).

RNA extractions were performed using the RNeasy kit (#74116, Qiagen, France) and QIAcube according to the manufacturer’s instructions (Qiagen, Paris, France). Reverse transcription was then performed with a QuantiTect Reverse Transcription Kit (#205311, Qiagen, Paris, France), and PCR was performed with a QuantiFast SYBR Green PCR Kit (#204156, Qiagen, Paris, France) and Rotor-Gene Q (Qiagen, Paris, France). Primers used were the QuantiTect Primers assay (#249900, Qiagen, Paris, France) and, more precisely, RPLP0 (QT00075012), KHDRBS3 (QT00065296), METTL3 (QT00036540), and HuR (QT00037856).

We performed real-time qPCR analysis (Bio-Rad; thermal cycler CFX96^TM) to evaluate mRNA levels of the target genes, RELA (p65), c-REL, bcl-2, TNF-α, Tp63, EGFR, STAT3, wnt5α and Tp53, and the reference housekeeping gene, hGAPDH, using specific primers for human genome (QuantiTect^® primers assay, Qiagen) (Table 1), and iQ^TM SYBR^® Green Supermix (BIO-RAD), as we previously described [10 (link)]. Assays were performed in 96 well-plates, in triplicate for each sample, and data were analyzed by CFX96 Manager^TM software. Relative mRNA expression levels were estimated for each target gene relative to reference gene (ΔΔC_T).