tissue culture methods and irradiation procedure have been described in . the three dimensional tissue culture model
( 3d epioral ) and media were purchased from mattek corporation ( ashland , ma ) .
the 3d epioral tissue is a co - culture organotypic model and consisted of human fibroblasts on the bottom and human oral keratinocytes grown on top of the fibroblasts . in order to induce differentiation and stratification of the keratinocytes of the 3d cultures ,
then the tissues were irradiated ( dose of irradiation was 0 , 2 , or 12  gy ) at the city of hope ( duarte , ca ) facility .
subsequently the tissues were incubated for 6  h at 37  c with 5% co2 after irradiation .
then one of the tissues was placed in 10% formalin for histopathological studies and the others were used for the extraction of total rna .
the rneasy plus mini kit ( qiagen , germantown , md ) was used to extract total rna of at least two identically treated tissues .
the integrity and quality of rna was determined by evaluating the a260/280 absorbance ratio using an agilent 2100 bioanalyzer ( agilent technologies , palo alto , ca ) . only rna samples with absorbance ratio , a260/280  >  2.0 , and rin  > 
the rna was converted to double - stranded cdna and amplified using in vitro transcription with t7 polymerase ; the transcription reaction included aminoallyl utp ( aa - dutp ) and the subsequent product was conjugated to cy5 nhs ester ( ge healthcare life sciences , pittsburg , pa ) . the human whole genome onearray v4.3 ( phalanx biotech , san diego , ca ) was used to perform the dna microarray analysis .
for this purpose , a quantity of 0.025  mg / ml fragmented cy5-labeled cdna was hybridized overnight at 42  c using the hybbag mixing system with 1   onearray hybridization buffer ( phalanx biotech ) and 0.01  mg / ml sheared salmon sperm dna ( promega , madison , wi ) . after the hybridization procedure
the raw cy5 intensity signals produced by each of the microarrays were captured using a molecular devices axon 4100a scanner and measured using genepix pro software and stored in gpr format .
the data from all the arrays of each experimental set were analyzed using rosetta resolver system ( rosetta biosoftware , usa ) .
testing was done in triplicate by combining technical replicates while the statistical analyses were done using the proprietary modeling techniques of rosetta resolver .
the error - weighted approach ( which is specifically geared towards combining replicated hybridizations to improve measurement precision and accuracy ) was used to calculate the average expression values . in order to decide whether intensity data was significantly above background , p - values
were calculated to test the null hypothesis that expression is absent ( called  p - value detected  ) . a p - value detected <  0.05 indicated that the transcript specific to a given probe was truly present or expressed .
lastly , we also calculated p - values for determining whether genes were differentially expressed . rather than focusing solely on fold changes ,
rosetta resolver uses error - model - based hypothesis tests , which account for both fold change and expression level .
three technical replicate hybridizations were averaged while ensuring that the technical replicates were of high repeatability . using the r function boxplot , raw and normalized log2 data from each sample were plotted but we did not include control and flagged probes .
this analysis not only helps finding hybridization with aberrant intensity distributions but also helps to confirm that the normalization of each replicate microarray has an appropriately centered distribution . for each sample
the scattered plots of raw and normalized log2 data were compared using the r function pairs .
pearson correlation tables were used to define the linear relationship of the two variables , raw and normalized data , in the scatter plots . for each technical repeat , we calculated correlation values for raw and normalized log2 intensities and we included in the calculation only probes with p - value of <  0.01 .
scatter plots showed that technical replicates had high repeatability and all the correlation values were >  0.953 .
our article , , emphasizes the differentially expressed genes of tissues that received different amounts of radiation ( i.e. 0 , 2 , or 12  gy ) , while herein we show the profiling of all the transcriptomic data resulting from these treatments .
we only used genes with |  fold change  |  >  1.5 and p - value  <  0.05 .
we input gene symbols into david bioinformatics , while throughout we used the default settings .
the threshold for significance was a benjamini - adjusted p - value  <  0.05 . based on our hypothesis , irradiated samples ( compared to non - irradiated control samples ) would display patterns of gene expression consistent with the physiological effects of irradiation .
table 2 includes selected enriched categories from our qc enrichment analysis ( table s1 contains all enrichment analysis results ) .
that is , up - regulated genes in irradiated samples were strongly enriched for functional categories involved in cell death , cell proliferation , oncogenesis , and ubiquitin conjugation .
also , down - regulated genes in irradiated samples were strongly enriched for functional categories involved in rna processing and cell division .
overall , these results confirmed the quality of the microarray data and facilitated further interpretation of the data presented in .
the following are the supplementary data related to this article.table s1enrichment analysis results using david bioinformatics .
up- and down - regulated genes were analyzed separately for each condition , so there are two separate worksheets listing enrichment analysis results .
goterm cc  =  gene ontology cellular component , mf  =  molecular function , bp  =  biological process , and up_seq_feature  =  uniprot sequence feature .
other enriched categories are from the interpro , sp pir , smart , and kegg databases .
count  =  the number differentially expressed genes annotated with a given enriched term .
adj p - val  =  benjamini - adjusted p - values . more information on the output from david bioinformatics can be found here : http://david.ncifcrf.gov/content.jsp?file  =  function_annotation.html .
up- and down - regulated genes were analyzed separately for each condition , so there are two separate worksheets listing enrichment analysis results .
goterm cc  =  gene ontology cellular component , mf  =  molecular function , bp  =  biological process , and up_seq_feature  =  uniprot sequence feature .
other enriched categories are from the interpro , sp pir , smart , and kegg databases .
count  =  the number differentially expressed genes annotated with a given enriched term .
more information on the output from david bioinformatics can be found here : http://david.ncifcrf.gov/content.jsp?file  =  function_annotation.html .
tissue culture methods and irradiation procedure have been described in . the three dimensional tissue culture model
( 3d epioral ) and media were purchased from mattek corporation ( ashland , ma ) .
the 3d epioral tissue is a co - culture organotypic model and consisted of human fibroblasts on the bottom and human oral keratinocytes grown on top of the fibroblasts . in order to induce differentiation and stratification of the keratinocytes of the 3d cultures ,
then the tissues were irradiated ( dose of irradiation was 0 , 2 , or 12  gy ) at the city of hope ( duarte , ca ) facility .
subsequently the tissues were incubated for 6  h at 37  c with 5% co2 after irradiation .
then one of the tissues was placed in 10% formalin for histopathological studies and the others were used for the extraction of total rna .
the rneasy plus mini kit ( qiagen , germantown , md ) was used to extract total rna of at least two identically treated tissues .
the integrity and quality of rna was determined by evaluating the a260/280 absorbance ratio using an agilent 2100 bioanalyzer ( agilent technologies , palo alto , ca ) . only rna samples with absorbance ratio , a260/280  >  2.0 , and
the rna was converted to double - stranded cdna and amplified using in vitro transcription with t7 polymerase ; the transcription reaction included aminoallyl utp ( aa - dutp ) and the subsequent product was conjugated to cy5 nhs ester ( ge healthcare life sciences , pittsburg , pa ) .
the human whole genome onearray v4.3 ( phalanx biotech , san diego , ca ) was used to perform the dna microarray analysis . for this purpose ,
a quantity of 0.025  mg / ml fragmented cy5-labeled cdna was hybridized overnight at 42  c using the hybbag mixing system with 1   onearray hybridization buffer ( phalanx biotech ) and 0.01  mg / ml sheared salmon sperm dna ( promega , madison , wi ) . after the hybridization procedure ,
the raw cy5 intensity signals produced by each of the microarrays were captured using a molecular devices axon 4100a scanner and measured using genepix pro software and stored in gpr format .
the data from all the arrays of each experimental set were analyzed using rosetta resolver system ( rosetta biosoftware , usa ) .
testing was done in triplicate by combining technical replicates while the statistical analyses were done using the proprietary modeling techniques of rosetta resolver .
the error - weighted approach ( which is specifically geared towards combining replicated hybridizations to improve measurement precision and accuracy ) was used to calculate the average expression values . in order to decide whether intensity data was significantly above background , p - values
were calculated to test the null hypothesis that expression is absent ( called  p - value detected  ) . a p - value detected <  0.05 indicated that the transcript specific to a given probe was truly present or expressed .
lastly , we also calculated p - values for determining whether genes were differentially expressed . rather than focusing solely on fold changes ,
rosetta resolver uses error - model - based hypothesis tests , which account for both fold change and expression level .
three technical replicate hybridizations were averaged while ensuring that the technical replicates were of high repeatability . using the r function boxplot , raw and normalized log2 data from each sample were plotted but we did not include control and flagged probes .
this analysis not only helps finding hybridization with aberrant intensity distributions but also helps to confirm that the normalization of each replicate microarray has an appropriately centered distribution . for each sample
the scattered plots of raw and normalized log2 data were compared using the r function pairs .
pearson correlation tables were used to define the linear relationship of the two variables , raw and normalized data , in the scatter plots . for each technical repeat , we calculated correlation values for raw and normalized log2 intensities and we included in the calculation only probes with p - value of <  0.01 .
scatter plots showed that technical replicates had high repeatability and all the correlation values were >  0.953 .
our article , , emphasizes the differentially expressed genes of tissues that received different amounts of radiation ( i.e. 0 , 2 , or 12  gy ) , while herein we show the profiling of all the transcriptomic data resulting from these treatments .
we only used genes with |  fold change  |  >  1.5 and p - value  <  0.05 .
we input gene symbols into david bioinformatics , while throughout we used the default settings .
the threshold for significance was a benjamini - adjusted p - value  <  0.05 . based on our hypothesis , irradiated samples ( compared to non - irradiated control samples ) would display patterns of gene expression consistent with the physiological effects of irradiation .
table 2 includes selected enriched categories from our qc enrichment analysis ( table s1 contains all enrichment analysis results ) .
that is , up - regulated genes in irradiated samples were strongly enriched for functional categories involved in cell death , cell proliferation , oncogenesis , and ubiquitin conjugation .
also , down - regulated genes in irradiated samples were strongly enriched for functional categories involved in rna processing and cell division .
overall , these results confirmed the quality of the microarray data and facilitated further interpretation of the data presented in .
the following are the supplementary data related to this article.table s1enrichment analysis results using david bioinformatics .
up- and down - regulated genes were analyzed separately for each condition , so there are two separate worksheets listing enrichment analysis results .
goterm cc  =  gene ontology cellular component , mf  =  molecular function , bp  =  biological process , and up_seq_feature  =  uniprot sequence feature .
other enriched categories are from the interpro , sp pir , smart , and kegg databases .
count  =  the number differentially expressed genes annotated with a given enriched term .
more information on the output from david bioinformatics can be found here : http://david.ncifcrf.gov/content.jsp?file  =  function_annotation.html .
up- and down - regulated genes were analyzed separately for each condition , so there are two separate worksheets listing enrichment analysis results .
goterm cc  =  gene ontology cellular component , mf  =  molecular function , bp  =  biological process , and up_seq_feature  =  uniprot sequence feature .
other enriched categories are from the interpro , sp pir , smart , and kegg databases .
count  =  the number differentially expressed genes annotated with a given enriched term .
more information on the output from david bioinformatics can be found here : http://david.ncifcrf.gov/content.jsp?file  =  function_annotation.html .
 we evaluated a three - dimensional ( 3d ) human oral cell culture that consisted of two types of cells , oral keratinocytes and fibroblasts as a model of oral mucositis which is a debilitating adverse effect of chemotherapy and radiation treatment . 
 the 3d cell culture model was irradiated with 12 or 2  gy , and total rna was collected 6  h after irradiation to compare global gene expression profiles via microarray analysis . here 
 we provide detailed methods and analysis on these microarray data , which have been deposited in gene expression omnibus ( geo ) : gse62395 . 
Direct link to deposited data
Experimental design, materials and methods
Tissue culture and irradiation
RNA isolation and microarray hybridization
Microarray data pre-processing and statistical analysis
Microarray data quality control
Conflict of interest