The cells were washed twice with cold PBS. Then 350 μl lysis buffer (1% β-mercapthanol in RLT buffer) was added to the mTOR kinase assay cells according to the protocol of Qiagen RNeasy® mini kit (Qiagen Benelux B.V.) after which the plate was stored at -80°C for later use. RNA isolation and reverse transcription mRNA was isolated from the gingival fibroblast lysates according to the manufacturer’s protocol of Qiagen RNeasy® mini kit (Qiagen Benelux B.V.). The mRNA concentrations of the samples were determined using the Nanodrop ND_1000 (Isogen Life Science). mRNA was reverse transcribed using the Fermentas first-strand cDNA synthesis
kit (Fermentas GmbH, St. Leon-Rot, Germany) according to the manufacturer’s protocol. Real-Time PCR cDNA synthesized from mRNA isolated from gingival fibroblasts after infection with P. gingivalis was analyzed in quadruple using Real-Time PCR with gene-specific primers on a ABI Prism 7000 Sequence Detecting System (Applied Biosystems, Nieuwerkerk a/d lJssel, The Netherlands). Reactions were performed with 2 ng cDNA in a total volume of 8 μl containing SYBR Green PCR Master Mix (Applied Biosystems)
and 0.99 pM of each primer. After activation Selleck MM-102 of the AmpliTaq Gold DNA polymerase for 10 minutes at 94°C, 40 cycles were run of a two step PCR consisting of a denaturation step at 95°C for 30 seconds and annealing and extension step at 60°C for 1 minute. Predicted product sizes were in the 100-200 bp range. Subsequently the PCR products were subjected to melting curve analysis to test if any unspecific PCR products were generated. The PCR reactions of the different amplicons had equal efficiencies. Samples were normalized for the selleck expression of housekeeping gene GAPDH, which is not affected by the experimental conditions, by calculating the Δ Ct (Ct housekeeping gene – Ct gene of interest) and expression of the different genes is expressed as 2-(ΔCt). Fold increase in gene expression (induction) was expressed by 2 -(ΔΔCt), wherein ΔΔCt = ΔCtchallenged- average Ct-value non-challenged. Statistical analysis
Differences in gene induction between multiple groups were tested by one-way analysis of variance (ANOVA) and Bonferroni’s Multiple Comparison Test. Tests were performed with GraphPad Prism version 4.00 for Windows, GraphPad Software, San Diego Meloxicam California USA. Differences were considered significant at p < 0.01. Acknowledgements We would like to thank Jeffrey Kroon for his excellent work on the transcriptional analysis of the P. gingivalis genes. Electronic supplementary material Additional file 1: Hydrophobicity of P. gingivalis strains. Percentage of bacterial cells adhered to hexadecane after extensive vortexing and 10 minutes incubation. 3.4%, 61% and 19% of the cells was adhered to hexadecane for W83, the epsC mutant and the complemented mutant respectively, indicating increased hydrophobicity for the epsC mutant. The data are the averages of two experiments comprised of triplicate measurements.