01). All DNA microarray work in this study was in compliance with MIAME guidelines and all data have been deposited under accession number E-TABM-467, in the ArrayExpress databases http://​www.​ebi.​ac.​uk/​arrayexpress. Validation of microarray data by real time, reverse transcription-PCR Total RNA (1

μg) was reverse transcribed to cDNA using SuperScript III First Strand Synthesis Supermix (MLN2238 molecular weight Invitrogen) in the presence of random primers (50 ng) according to the manufacturer’s recommendations. Real time-PCR was carried out using a Rotor-Gene 3000 (Corbett Research, Sydney, Australia). The primers for the real-time analysis (Table 1) were designed using Primer3 software http://​primer3.​sourceforge.​net/​. The lengths of the primers were 18 to 20 nucleotides and the amplified products between PLK inhibitor www.selleckchem.com/products/EX-527.html 109 and 130-bp. The amplification efficiency of each primer set was determined empirically by using cDNA template dilutions over four orders of magnitude. The amplification efficiency for each primer set varied between 95.4% and 106.6%, showing that the amplicons were generated with comparable efficiency. Table 1 Primers used for real-time reverse transcription PCR Gene ID Forward

primer 5′-3′ Reverse primer 5′-3′ PG0158 TTCTTTTGGTGGACGATGTG GAGGGACGCTTGGTAACG PG0270 TCGCAAGCCAAGCAAATAC GAGATAGGGTGCGATGGTTG PG0347 TCGGCGATGACTACGACA CGCTCGCTTTCTCTTCATTC PG0553 CCGATGGCAATACGAGCCGC ATAGCCGGGGCACAGAGGGC PG0593 CAAAAGGTCGCTCCACTCA GTTCGCCACGATCATTCAC PG0914 TCATCGCTCGCAGTAAGAAC CTGAATACCGAATCCCCATC PG1055 AGCCAACAGGAGATGGAGTG TCAAGTCGGAGTGCGAAAA PG1431 CGCAGACCAATCGCATAAG

CAGAATAGCCATCGCACAGA PG1432 CCATGCAGCAAGGAGATACA TAGTGTCGAGGGCCATTTTC The real time-PCR reaction contained 12.5 μL of Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen), 0.2 μM of each gene-specific primer and 5 μL of cDNA template. The cycling conditions were 50°C for 2 min, 95°C for 2 min, then 40 cycles of 95°C for 15 s, 58°C for 30 s, and 72°C for 30 s. Negative controls of distilled water and total RNA samples were included in each run. All reactions were carried out in triplicate and melting curve analysis indicated that in each reaction a single product was amplified. PG0347 encoding a putative UDP-glucose 4-epimerase, galE, was selected as normalizer for all reactions. The critical threshold cycle, CT for each gene was generated by the Rotor-Gene 6 software (Corbett Interleukin-2 receptor Research) and the relative expression ratio of the selected genes calculated and analyzed using the relative expression software tool (REST) http://​www.​gene-quantification.​info[23]. Each real time-PCR reaction was performed using the biological replicate total RNA samples that were used for microarray analysis. Results and Discussion P. gingivalis W50 growth in continuous culture and biofilm formation P. gingivalis is a slow growing anaerobe that even in rich media has a generation time of 4.65 h [24]. In the continuous culture system we employed here P.