P. gingivalis strains were grown on Fastidious anaerobe (+5% Horse blood) for 48h: Invasive samples were recovered from OSCC cell lines after invasion in antibiotic protection assays according to Suwannakul et al., 2010 (Microbiology, Jun 24.), non-invasive cells were recovered from cell supernatants also as described in Suwannakul et al.,. Similarly High and low protease samples represent low and high protease isolates grown on FA-blood for 48h. all anaerobically at 37C
Extracted molecule
total RNA
Extraction protocol
RNA samples used in microarray studies were extracted, purified and quantified as described earlier (section 2.4.2.1) All RNA samples exhibited A260/A280 ratio of at least 1.8 and the concentration was > least 3 mg/ml. RNA samples were checked by RNA gel electrophoresis.
Label
cy3
Label protocol
The cDNA was generated using random primers for reverse transcription (Invitrogen). The reverse transcription reaction comprised 16 μg of P.gingivalis , 6 μg of random primer hexamers, the first strand buffer (75 mM KCl, 50 mM Tris-HCl (pH 8.3), 3 mM Mgcl2, 12.5 mM dATP, dCTP, and dGTP; 4.16 mM dTTP; and 8.33 mM amino allyl-dUTP, 800 units of Superscript II and nuclease free water (Qiagen) in a 30 μl final volume in a PCR reaction tube (All reagents in the reverse transcriptase reaction were purchased from the Invitrogen,UK except amino allyl-dUTP which was purchased from Sigma ). The reaction was mixed by inversion. For cDNA generation the randomized primers were annealed at 70°C for 10 min followed by snap-freezing in a dry ice/ethanol bath for 30 sec and centrifugation for 1 min. The reaction mixture (Superscript II buffer, 0.1M DTT, and aa-dNTP mix) was then incubated with Superscript II reverse transcriptase (Invitrogen) at 42°C overnight in a DNA Engine DYAD TM thermal cycler. Residual RNA was removed by alkaline treatment followed by neutralization (The RNA was hydrolyzed by incubation with 10 μl of 0.5 M EDTA and 10 μl 1 M NaOH at 65 °C for 15 min. The sample was neutralized by 1M HCL before purification of the cDNA). The cDNA was purified using a QIAquick PCR purification kit (Qiagen) according to the manufacturers instruction except that the phosphate wash and elution buffers (see details of preparation in section 2.4.1.2) were substituted for the Qiagen supplied buffers because the Qiagen buffers contain free amines, which compete with the Cy dye coupling reaction). Purified cDNAs were labelled with 4.5 μl N-hydroxysuccinimide-Cy3 dye (40 nmol) dissolved in dimethyl sulfoxide (Amersham Biosciences) in the presence of 4.5 μl 0.1 M Na2CO3 buffer, pH 9.0. These cDNA samples were allowed to incubate at room temperature in the dark for 60 min. After incubation, the labelling mixtures were cleaned again using the QIAquick PCR purification Kit (Qiagen) according to the manufacturers instruction to remove uncouple Cy-dye. These cDNAs were then speed dried for 30 min.
Hybridization protocol
The labelled cDNA probe was applied to pre-hybridised P.gingivalis whole genomic microarray slides (Customized by TIGR, USA). The microarray slides were pre-hybridised with pre-hybridisation buffer (detail was described in section 2.5.1.3). Briefly, the array slide was thoroughly washed with distilled water (room temperature), snapped dried on a heat block for 2-3 seconds and UV cross-linked at 250 mJoules. Slides were then hydrated by dipping in pre-warmed 42°C water, centrifuged (7000 g, 5 min) at room temperature to dry the slide and then incubated in a 50-mL coplin jar containing pre-warmed pre-hybridisation buffer for at least 45 min at 42°C. When the hybridisation reaction was ready, the array slide was prepared by dipping in pre-warmed water (42°C) and agitating 2-3 min to remove pre-hybridisation buffer. The array slide was rinsed in isopropanol and centrifuged 5 for min (7000 g, 5 min) at room temperature to dry. The cDNA-labelling probe was prepared for array hybridisation by adding 40 μl of hybridisation buffer (detail as described in section 2.5.1.4) and gently pipetting up and down. The hybridizing probe mixture was then boiled for 5 min, centrifuged (10,000 x g, 5 min), and cooled for 2 min at room temperature. The probe mixture was applied gently to the array slide, carefully covered with a square cover slip (Nugene), and placed in a hybridisation chamber in a water bath (42°C) and incubated 18 h in the dark. After hybridisation, the slides were removed from the chamber taking care not to disturb the cover slip. The cover slip was removed by submerging the slide with the cover slips facing down in a dish containing warm 1x SSC, 0.05 % SDS wash buffer. The cover slips were dislodged by gently moving the slides from side-to-side and the slides placed in a rack in a fresh bath with warm (65°C) 1xSSC 0.05% SDS (First washing buffer, see details in 2.5.1.5). The array slide was agitated for 2-3 min, transferred to a second bath of buffer and re-agitated for 2-3 min and repeated for a third and fourth times (details in section 2.5.1.5 were used to wash). The slides were then blotted on paper towels to remove as much SDS-containing buffer as possible, then transferred to a fresh solution of 0.06 x SSC and agitated for 2-3 min. Finally the slides were centrifuged (700x g, 5 min) at room temperature to dry them and were kept in the dark until scanned.
Scan protocol
The array slide was scanned using a GenePix scanner (supplied by Prof. Robert Poole Department of Molecular Biology and Biotechnology, University of Sheffield) at 532nm (Cy3 channel) and the images were saved as tif picture files prior to analysis.
Description
SAMPLE 13
Data processing
Data from six individual experiments were normalized and then analyzed using the Spotfinder Software (TM4, The Institute of Genomic Research; TIGR, www.tigr.org). The data points with a density below 100,000 were discarded from analysis. A cut off ratio of 1.5:1 was used on all the slides. Significant Analysis of Microarray Software (SAM; TM4, and TIGR) was used to test statistical significance of the results from the microarray experiments. This statistical analysis involved sorting the change in expression of each gene relative to the standard deviation of all replicates for that gene. Therefore, genes with even a small change were not discounted if the ratios were consistent among all the repeats, thus effectively reducing false-negatives. False-positives were avoided when genes had poor reproducibility between replicates. Spot intensities for all channels were entered into SAM as paired, unlogged values. Delta values were chosen according to the lowest false discovery rate, which for this study was 4.7%. In combination with the 1.5 fold change, the genes with expression ratios of ≥3.5 were considered biologically significant.
