The paper points were incubated in water at 37 °C with shaking for 24 h. After the paper points were removed, the DNA was amplified by PCR (GoTaq Polymerase; Promega, Madison, WI) with primers specific for the 16S RNA gene. The sequences were 5′-TGGGTTTAAAGGGTGCGTAG-3′ for the forward primer and 5′-CAATCGGAGTTCCTCGTGAT-3′ for the reverse primer (Meuric et al., 2008). After HistoGene staining, sections were microdissected

using a Veritas LCM system (Molecular Devices). For each sample, approximately 1 mm2 of each site of interest was microdissected with learn more a separated ‘cap’ (Capsure Macro LCM Caps; Molecular Devices, Arcturus). Three main gingival tissue structures were microdissected (Fig. 1): epithelium, connective tissue without infiltrates, and inflammatory infiltrates in connective tissue. RNA was extracted

from microdissected sections with the PicoPure RNA isolation kit (Molecular Devices) according to the manufacturer’s protocol. Total RNA extract was eluted in 30 μL of water. RNA from cultured P. gingivalis (ATCC 33277) was extracted with the RNAeasy mini kit (Qiagen, Hilden, Germany) and used as a positive control. For all samples, the quantity and quality of RNA were measured with the Nanodrop 1000 (Nanodrop, Wilmington, DE). Reverse transcription (RT) was performed using M-MLV transcriptase (Promega, Madison, WI) according to the manufacturer’s protocol. For Selleckchem Saracatinib each microdissected sample, an RT reaction without reverse transcriptase was performed to check for the presence of genomic DNA. Primers from

Meuric et al. (Meuric et al., 2008) were used to detect P. gingivalis Meloxicam 16S RNA. Quantitative PCR was performed using the qPCR Master Mix Plus for Sybr Green I (Eurogentec, Liege, Belgium), 2 μL of cDNA, and 0.4 μM primer. For each sample, measurements of the 16S RNA gene were taken in triplicate. Threshold cycle (Ct) values were converted into number of bacteria (normalized for 1 ng of total RNA) by comparison with a standard curve constructed using serial dilutions of cDNA from P. gingivalis 33277. A P value was determined to compare epithelium and connective tissue with or without inflammatory infiltrates for each biopsy sample using the analysis of variance (anova) test (ezanova software). Monoclonal mouse antibodies against human CD3, CD138, CD14, CD5, CD27, CD4, and CD8 were obtained from Beckman Coulter (Villepinte, France), and goat anti-CD20 antibody was obtained from Neomarkers (Fremont, CA). Rabbit anti-P. gingivalis ATCC 33277 was produced in our laboratory by injection of P. gingivalis 33277 whole-cell extract. Secondary antibodies used for this study were fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat or anti-rabbit antibody (Jackson Immuno-Research, West Grove, PA) and tetramethylrhodamine isothiocyanate (TRITC)-conjugated donkey anti-mouse or anti-goat antibody (Jackson ImmunoResearch).