After careful removal of supra-gingival plaque, the curette
was placed subgingivally until the bottom MM-102 chemical structure of the probeable pocket was reached and subgingival plaque was collected by a single scaling stroke. The individual plaque samples were transferred into Eppendorf tubes containing 200 μl of sterile T-E buffer (10 mM Tris HCl, 1.0 mM EDTA, pH 7.6) and were not pooled at any stage of the processing described below. Processing of plaque samples Immediately after transfer to the laboratory the plaque pellet was re-suspended, vigorously vortexed, and 200 μl of a 0.5 M NaOH solution were added. Digoxigenin-labeled, whole genomic probes were prepared by random priming by the use of the High-Prime labeling kit (Roche/Boehringer-Mannheim, Indianapolis, IN, USA) from the following microbial strains: Aggregatibacter actinomycetemcomitans (ATCC 43718), Porphyromonas gingivalis (ATCC 33277), Tannerella forsythia (ATCC 43037), Treponema denticola (ATCC 35404), Prevotella intermedia (ATCC 25611), Fusobacterium nucleatum (ATCC 10953), Parvimonas micra (ATCC 33270), Campylobacter rectus (ATCC 33238), Eikenella corrodens (ATCC 23834), Veillonella parvula (ATCC 10790), and Actinomyces naeslundii (ATCC 49340). Further processing was carried out according to the checkerboard {Selleck Anti-cancer Compound Library|Selleck Anticancer Compound Library|Selleck Anti-cancer Compound Library|Selleck Anticancer Compound Library|Selleckchem Anti-cancer Compound Library|Selleckchem Anticancer Compound Library|Selleckchem Anti-cancer Compound Library|Selleckchem Anticancer Compound Library|Anti-cancer Compound Library|Anticancer Compound Library|Anti-cancer Compound Library|Anticancer Compound Library|Anti-cancer Compound Library|Anticancer Compound Library|Anti-cancer Compound Library|Anticancer Compound Library|Anti-cancer Compound Library|Anticancer Compound Library|Anti-cancer Compound Library|Anticancer Compound Library|Anti-cancer Compound Library|Anticancer Compound Library|Anti-cancer Compound Library|Anticancer Compound Library|Anti-cancer Compound Library|Anticancer Compound Library|buy Anti-cancer Compound Library|Anti-cancer Compound Library ic50|Anti-cancer Compound Library price|Anti-cancer Compound Library cost|Anti-cancer Compound Library solubility dmso|Anti-cancer Compound Library purchase|Anti-cancer Compound Library manufacturer|Anti-cancer Compound Library research buy|Anti-cancer Compound Library order|Anti-cancer Compound Library mouse|Anti-cancer Compound Library chemical structure|Anti-cancer Compound Library mw|Anti-cancer Compound Library molecular weight|Anti-cancer Compound Library datasheet|Anti-cancer Compound Library supplier|Anti-cancer Compound Library in vitro|Anti-cancer Compound Library cell line|Anti-cancer Compound Library concentration|Anti-cancer Compound Library nmr|Anti-cancer Compound Library in vivo|Anti-cancer Compound Library clinical trial|Anti-cancer Compound Library cell assay|Anti-cancer Compound Library screening|Anti-cancer Compound Library high throughput|buy Anticancer Compound Library|Anticancer Compound Library ic50|Anticancer Compound Library price|Anticancer Compound Library cost|Anticancer Compound Library solubility dmso|Anticancer Compound Library purchase|Anticancer Compound Library manufacturer|Anticancer Compound Library research buy|Anticancer Compound Library order|Anticancer Compound Library chemical structure|Anticancer Compound Library datasheet|Anticancer Compound Library supplier|Anticancer Compound Library in vitro|Anticancer Compound Library cell line|Anticancer Compound Library concentration|Anticancer Compound Library clinical trial|Anticancer Compound Library cell assay|Anticancer Compound Library screening|Anticancer Compound Library high throughput|Anti-cancer Compound high throughput screening| DNA-DNA hybridization method [26] as earlier described [27] with
the following modifications: The chemiluminescent substrate used for detection was CSPD (Roche/Boehringer-Mannheim). Evaluation of the chemiluminescence signal was performed in a LumiImager F1 Workstation (Roche/Boehringer-Mannheim) by comparing the obtained signals with the ones Torin 2 generated by pooled standard samples containing 106 or 105 of each of the species. Standard curves were generated for each
species by means of the LumiAnalyst software (Roche/Boehringer-Mannheim), and the obtained chemiluminescent signals were ultimately transformed into bacterial counts and exported into Excel files. Statistical Analysis In all analyses, either R version 2.3.1 (Linux OS) or SAS for PC version 9.1 (SAS Institute, Cary, NC) were used. Gene expression data Rebamipide were normalized and summarized using the log scale robust multi-array analysis (RMA, [28]) with default settings. Laboratory analysis provided a relative quantity of individual bacterial species for each plaque sample by comparison to known standards. Because the distribution of absolute bacterial counts was skewed, values were natural logarithm (ln) transformed, averaged within mouth and standardized by dividing each respective ln(bacterial count) by the population standard deviation for the respective species: one standard deviation on the ln scale (SDln) was treated as equivalent across microbes as previously described [29].