all the samples were collected from patients undergoing surgical ablation of larynx squamous cell carcinoma at the head and neck surgery division of the department of ophthalmology , otorhinolaryngology and head & neck of ribeirao preto medical school , usp ( brazil ) . inclusion criteria were : histopathological diagnosis of lscc , elective surgeries for lscc in patients without previous treatments and patients ' allowance to donate part of their tumor for genetic studies . exclusion criteria were : doubtful diagnosis of lscc , unavailable post - surgical follow - up , patients without complete clinical data or signed agreement for collection of samples . a total of twenty nine lscc tumor samples and thirteen adjacent non - neoplastic tissues proceed for the transcriptome analysis . clinical information and tnm staging classification of the lscc patients most of the patients were male ( 96.8% ) , all of them were smokers and alcoholic , including both current and former . thirty five percent of the tumors were originated from glottis , followed by larynx ( 29.0% ) and supraglottis ( 22.6% ) . patients were further classified according to the tnm system : 48.4% of the patients were in early staging and 51.6% in late staging . this study was approved by the ethics committee of ribeirao preto medical school , university of sao paulo ( usp ) ( proc . after lscc histopathological confirmation and microdissection of the tumors from their non - neoplastic adjacent tissue , the samples were store in liquid nitrogen . after extraction , the rna was purified with rneasy kit ( qiagen , usa ) and quantified with nanodrop spectrophotometer ( thermo scientific , usa ) . its quality was evaluated by 1.5% agarose gel electrophoresis ( 28s and 18s ribosomal rna detection ) . for the microarray analysis it was used the whole human genome oligo microarray kit 4    44k ( agilent , g4112f , usa ) . cdna was hybridized to the microarray chip in the fluidics station 450 system ( affymetrix , usa ) , using the quick amp labeling one - color kit ( agilent , usa ) . as an internal mrna control , the one color rna spike - in kit was used ( agilent , usa ) . agilent feature extraction software ( version 9.5.3.1 ) was used to analyze acquired array images . plain text files were loaded and processed into r with the bioconductor package agi4    44pre - process . the cv.rep.probes function was used to estimate the percent of coefficient of variation ( % cv ) for replicated non - control probes , inferring the reproducibility of the arrays ( fig . the gprocessedsignal and the gbgused values of each array were loaded as foreground and background signals , respectively into an rglist object . the following flags were used to filter probes : control , well above bg , is found , well above neg ctrls , is saturated , population outlier , and non uniform outlier . additionally probe signals with at least 90% of good features in an experimental condition were selected . it was performed by looking for arrays ( a and b ) , which the sum ( s ) of the distances ( d ) to all other arrays , sa  =  b 
 dab was exceptionally large ; calculating the kolmogorov smirnov statistic ( ka ) between each array and the pooled data distribution ; and computing hoeffding 's statistic ( da ) on the joint distribution of a and m for each array ( m  =  log2(i1 )    log2(i2 ) ; a  =  1/2 ( log2(i1 )  +  log2(i2 ) ) , where i1 is the intensity of the array studied , and i2 is the intensity of a  pseudo-array that consists of the median across arrays ) . stagewise normalization is recommended when data include technical and biological replicates producing a better median chip . then , quantile normalization methods were applied , within groups and subsequently between all arrays . each set of replicated non - control probes has been collapsed into a single value computed as the median of the probes intensities belonging to the same set . principal component analysis ( pca ) was applied to classify samples by tumor and adjacent non - neoplastic tissue as demonstrated on fig . we compared the gene expression between tumor and adjacent non - neoplastic tissue using a non - paired - t test analysis . false discovery rate ( fdr ) adjusted p - value  <  1e    07 and fold change ( fc )  >  10 were accepted to consider genes to be differentially expressed , identifying a total of 81 probes above this cut - off ( fig . 3 ) . then we investigated the biological process of upregulated and downregulated genes in tumor compared to normal samples with web - based gene set analysis toolkit ( webgestalt ) . p - value was calculated using hypergeometric distribution and fdr as multiple hypothesis test correction method . tissue morphogenesis ( go : 0009888 ) and differentiation ( go : 0032332 ) are the main biological process in upregulated genes while cytokines and growth factor genes are highly expressed in adjacent non - neoplastic patients , suggesting a role on lscc tumorigenesis . all the samples were collected from patients undergoing surgical ablation of larynx squamous cell carcinoma at the head and neck surgery division of the department of ophthalmology , otorhinolaryngology and head & neck of ribeirao preto medical school , usp ( brazil ) . inclusion criteria were : histopathological diagnosis of lscc , elective surgeries for lscc in patients without previous treatments and patients ' allowance to donate part of their tumor for genetic studies . exclusion criteria were : doubtful diagnosis of lscc , unavailable post - surgical follow - up , patients without complete clinical data or signed agreement for collection of samples . a total of twenty nine lscc tumor samples and thirteen adjacent non - neoplastic tissues proceed for the transcriptome analysis . clinical information and tnm staging classification of the lscc patients most of the patients were male ( 96.8% ) , all of them were smokers and alcoholic , including both current and former . thirty five percent of the tumors were originated from glottis , followed by larynx ( 29.0% ) and supraglottis ( 22.6% ) . patients were further classified according to the tnm system : 48.4% of the patients were in early staging and 51.6% in late staging . this study was approved by the ethics committee of ribeirao preto medical school , university of sao paulo ( usp ) ( proc . after lscc histopathological confirmation and microdissection of the tumors from their non - neoplastic adjacent tissue , the samples were store in liquid nitrogen . after extraction , the rna was purified with rneasy kit ( qiagen , usa ) and quantified with nanodrop spectrophotometer ( thermo scientific , usa ) . its quality was evaluated by 1.5% agarose gel electrophoresis ( 28s and 18s ribosomal rna detection ) . for the microarray analysis it was used the whole human genome oligo microarray kit 4    44k ( agilent , g4112f , usa ) . cdna was hybridized to the microarray chip in the fluidics station 450 system ( affymetrix , usa ) , using the quick amp labeling one - color kit ( agilent , usa ) . as an internal mrna control , the one color rna spike - in kit was used ( agilent , usa ) . agilent feature extraction software ( version 9.5.3.1 ) was used to analyze acquired array images . plain text files were loaded and processed into r with the bioconductor package agi4    44pre - process . the cv.rep.probes function was used to estimate the percent of coefficient of variation ( % cv ) for replicated non - control probes , inferring the reproducibility of the arrays ( fig . the gprocessedsignal and the gbgused values of each array were loaded as foreground and background signals , respectively into an rglist object . the following flags were used to filter probes : control , well above bg , is found , well above neg ctrls , is saturated , population outlier , and non uniform outlier . additionally probe signals with at least 90% of good features in an experimental condition were selected . it was performed by looking for arrays ( a and b ) , which the sum ( s ) of the distances ( d ) to all other arrays , sa  =  b 
 dab was exceptionally large ; calculating the kolmogorov smirnov statistic ( ka ) between each array and the pooled data distribution ; and computing hoeffding 's statistic ( da ) on the joint distribution of a and m for each array ( m  =  log2(i1 )    log2(i2 ) ; a  =  1/2 ( log2(i1 )  +  log2(i2 ) ) , where i1 is the intensity of the array studied , and i2 is the intensity of a stagewise normalization is recommended when data include technical and biological replicates producing a better median chip . then , quantile normalization methods were applied , within groups and subsequently between all arrays . each set of replicated non - control probes has been collapsed into a single value computed as the median of the probes intensities belonging to the same set . principal component analysis ( pca ) was applied to classify samples by tumor and adjacent non - neoplastic tissue as demonstrated on fig . 2 . we compared the gene expression between tumor and adjacent non - neoplastic tissue using a non - paired - t test analysis . false discovery rate ( fdr ) adjusted p - value  <  1e    07 and fold change ( fc )  >  10 were accepted to consider genes to be differentially expressed , identifying a total of 81 probes above this cut - off ( fig . then we investigated the biological process of upregulated and downregulated genes in tumor compared to normal samples with web - based gene set analysis toolkit ( webgestalt ) . p - value was calculated using hypergeometric distribution and fdr as multiple hypothesis test correction method . tissue morphogenesis ( go : 0009888 ) and differentiation ( go : 0032332 ) are the main biological process in upregulated genes while cytokines and growth factor genes are highly expressed in adjacent non - neoplastic patients , suggesting a role on lscc tumorigenesis . we describe detailed technical methods to reproduce the analysis of whole human genome oligo microarray kit ( agilent , g4112f , usa ) from twenty - nine lscc tumor samples and thirteen adjacent non - neoplastic tissues . lscc is the second most common malignancies of the head and neck tumors , and this high frequency may be associated with the lifestyle of patients , , . the high quality gene expression data are concordant with important clinical parameters , demonstrating the relevant expression profile signature of differentially expressed genes . the following is the supplementary data related to this article.supplemental table 1tumor sample description . tumor sample description