H a tooth site demonstrating a probing depth 6 mm, clinical attachment level 5 mm and bleeding on probing were included in the periodontitis-affected group, according to the clinical parameters previously used as indicators of periodontitis [15,16,17]. During flap surgery, two ML 240 site adjacent gingival biopsies with identical clinical status were harvested from a periodontal pocket affected by periodontitis. The sizes of the specimens were approximately 262 mm, and included the connective tissue and the epithelium. In the same subjects, two adjacent gingival biopsies with identical clinical status and of about the same size were also obtained from a clinically healthy gingival pocket. Clinically healthy pockets were defined as sites with no gingival/periodontal inflammation, no bleeding on probing, a probing depth #3.5 mm and a clinical attachment level #3.5 mm. One 
of the biopsies from each site was stored in RNA Later (Applied Biosystems, USA) overnight at 4uC and thereafter stored at 280uC for subsequent RNA isolation. The second biopsy from each site was used for histological and immunohistochemical analysis.Hematoxylin-Eosin stainingDeparaffinized serial sections of gingival tissues were formalin fixed (4 neutral buffered formalin) and paraffin embedded. For assessment of orientation of the epithelium and connective tissue as well as the degree of inflammation, deparaffinized serial sections (4 mm) were prepared and sections of each biopsy were stained with Hematoxylin-Eosin (H E). The degree of inflammatory cell infiltration was evaluated by three blinded observers, using a relative scale from 0 to 3, and statistical differences between periodontitis-affected and healthy sites were tested using the Wilcoxon signed-rank test.0 = no evidence of inflammatory infiltration, 1 = slight inflammatory infiltration, 
2 = moderate inflammatory infiltration and 3 = severe inflammatory infiltration. 0 = no CD3 positive cells, 1 = low amount of CD3 positive cells, 2 = moderate amount of CD3 positive cells, 3 = high amount of CD3 positive cells and ?= not enough material to perform staining. c Significant difference between periodontitis-affected and healthy sites (p,0.01). d Significant difference between periodontitis-affected and healthy sites (p,0.05). doi:10.1371/journal.pone.0046440.tbHealthy sitesDespite research investigating periodontitis gene expression profiles through microarray analysis, specific genes responsible for the disease have not yet been found. However, the recent development of massively parallel sequencing has provided a more comprehensive and accurate tool for gene expression analysis through sequenced based assays of transcriptomes, RNA-Sequencing (RNA-Seq). This method enables analysis of the complexity of whole eukaryotic transcriptomes [12] and studies comparing RNA-Seq and microarrays have shown that RNA-Seq has less bias, a greater dynamic range, a lower frequency of false positive signals and higher reproducibility [13,14]. The aim of the present study was to investigate the general pattern of the gene expression profile in periodontitis using RNA-Seq. We also aimed to investigate the local variation in gene expression at site level, comparing periodontitis-affected and healthy gingival tissues obtained from the same patient.JI 101 site inflammation CD3 (0?)b, d1 1 2 1 3 8 42 MInflammation H E (0?)a, cProbing depth (mm)Inflammation CD3 (0?)b, dPeriodontitis-affected sitesInflammationH E (0?)a,cTable 1. Patient characteristics and.H a tooth site demonstrating a probing depth 6 mm, clinical attachment level 5 mm and bleeding on probing were included in the periodontitis-affected group, according to the clinical parameters previously used as indicators of periodontitis [15,16,17]. During flap surgery, two adjacent gingival biopsies with identical clinical status were harvested from a periodontal pocket affected by periodontitis. The sizes of the specimens were approximately 262 mm, and included the connective tissue and the epithelium. In the same subjects, two adjacent gingival biopsies with identical clinical status and of about the same size were also obtained from a clinically healthy gingival pocket. Clinically healthy pockets were defined as sites with no gingival/periodontal inflammation, no bleeding on probing, a probing depth #3.5 mm and a clinical attachment level #3.5 mm. One of the biopsies from each site was stored in RNA Later (Applied Biosystems, USA) overnight at 4uC and thereafter stored at 280uC for subsequent RNA isolation. The second biopsy from each site was used for histological and immunohistochemical analysis.Hematoxylin-Eosin stainingDeparaffinized serial sections of gingival tissues were formalin fixed (4 neutral buffered formalin) and paraffin embedded. For assessment of orientation of the epithelium and connective tissue as well as the degree of inflammation, deparaffinized serial sections (4 mm) were prepared and sections of each biopsy were stained with Hematoxylin-Eosin (H E). The degree of inflammatory cell infiltration was evaluated by three blinded observers, using a relative scale from 0 to 3, and statistical differences between periodontitis-affected and healthy sites were tested using the Wilcoxon signed-rank test.0 = no evidence of inflammatory infiltration, 1 = slight inflammatory infiltration, 2 = moderate inflammatory infiltration and 3 = severe inflammatory infiltration. 0 = no CD3 positive cells, 1 = low amount of CD3 positive cells, 2 = moderate amount of CD3 positive cells, 3 = high amount of CD3 positive cells and ?= not enough material to perform staining. c Significant difference between periodontitis-affected and healthy sites (p,0.01). d Significant difference between periodontitis-affected and healthy sites (p,0.05). doi:10.1371/journal.pone.0046440.tbHealthy sitesDespite research investigating periodontitis gene expression profiles through microarray analysis, specific genes responsible for the disease have not yet been found. However, the recent development of massively parallel sequencing has provided a more comprehensive and accurate tool for gene expression analysis through sequenced based assays of transcriptomes, RNA-Sequencing (RNA-Seq). This method enables analysis of the complexity of whole eukaryotic transcriptomes [12] and studies comparing RNA-Seq and microarrays have shown that RNA-Seq has less bias, a greater dynamic range, a lower frequency of false positive signals and higher reproducibility [13,14]. The aim of the present study was to investigate the general pattern of the gene expression profile in periodontitis using RNA-Seq. We also aimed to investigate the local variation in gene expression at site level, comparing periodontitis-affected and healthy gingival tissues obtained from the same patient.Inflammation CD3 (0?)b, d1 1 2 1 3 8 42 MInflammation H E (0?)a, cProbing depth (mm)Inflammation CD3 (0?)b, dPeriodontitis-affected sitesInflammationH E (0?)a,cTable 1. Patient characteristics and.
