RNA Isolation and qRT-PCR Analysis

For RNA isolation 2–3 mm tissue slices were collected and lysed in 1 mL TRIzol^® Reagent (Ambion^®, Life Technologies^TM, Carlsbad, CA, USA), and RNA was isolated as instructed by the manufacturer. RNA concentrations were determined by fluorometric measurements (Qubit, Invitrogen, Darmstadt, Germany), and RNA quality and integrity were identified by Qubit RNA IQ kit (Invitrogen). The proportion of intact RNA of total RNA isolates was at least 70%. Two micrograms of total RNA were reverse transcribed by M-MuLV Reverse Transcriptase with 2 micrograms of oligo dT₁₅ (GeneON, Ludwigshafen am Rhein, Germany) in a ThermoQ heating and cooling block (Biozym, Hessisch Oldendorf, Germany).
Specific sequences of primers used are detailed in Supplementary Table S1. The primers were synthesized by Invitrogen, Darmstadt, Germany and were used for real-time PCR utilizing the Sensifast Sybr Fluorescein Kit of Bioline (Labconsulting, Vienna, Austria) and the Bio-Rad MyiQ^TM (Bio-Rad, Laboratories, Inc., Hercules, CA, USA) cycler according to the manufacturer’s protocol.
GAPDH was used as housekeeping gene, and relative quantities of SNAI1, SLUG, ZEB1, TWIST and KLF4 transcripts were calculated by pair-wise differences of threshold cycles (∆_CT) of gene of interest and the loading control housekeeping gene [25 (link)]. According to Livak et al. [25 (link)], in the final analysis, we used the relative quantification and related the PCR signal in both HNSCC and control mucosa to a reference, which was the mean value of the control uvula samples from UPPP. The identity of the PCR products of genes discussed in this study were confirmed by Sanger sequencing by Microsynth Austria (Vienna, Austria).