After local anesthesia, submarginal incisions were performed, mucoperiosteal flaps were reflected, and the portion of each interproximal gingival papilla that adhered to the root surface was carefully dissected. This section comprised the epithelial lining of the interproximal periodontal pockets and the underlying connective tissue. After dissection, the gingival tissue specimens were ROCK inhibitor thoroughly rinsed with sterile normal saline solution and transferred into Eppendorf tubes containing a liquid RNA stabilization reagent (RNAlater, Ambion, Austin, TX). A minimum of 2 diseased papillae were harvested from each sextant

and, whenever available, a healthy tissue specimen was obtained from an adjacent site. After collection of the specimens, pocket elimination/reduction periodontal surgery was completed according to standard procedures. All patients received additional periodontal therapy according to their OSI 906 individual needs. RNA extraction, reverse transcription, in vitro cRNA synthesis The tissue specimens were stored in a liquid RNA stabilization reagent (RNAlater) overnight at 4°C, snap-frozen and stored in liquid nitrogen. All further processing occurred simultaneously LCZ696 price for gingival biopsies originating

from the same donor. Specimens were homogenized in Trizol (Invitrogen Life Technologies, Carlsbad, CA, USA). After incubation with chloroform and centrifugation at 12,000 g, RNA collected in the upper aqueous phase was precipitated by mixing with

75% isopropyl-alcohol and additional centrifugation and washings. The extracted RNA was purified using a total RNA isolation kit (RNeasy; Qiagen, Valencia, Paclitaxel CA, USA), quantified spectrophotometrically, and 7.5 micrograms of total RNA were reverse-transcribed using a one-cycle cDNA synthesis kit (GeneChip Expression 3′ amplification one-cycle cDNA synthesis kit; Affymetrix, Santa Clara, CA, USA). Synthesis of biotin-Labeled cRNA was performed using appropriate amplification reagents for in vitro transcription (GeneChip Expression 3′-Amplification Reagents for IVT labeling kit; Affymetrix). The cRNA yield was determined spectrophotometrically at 260 nm. Twenty μg of cRNA were fragmented by incubation in fragmentation buffer at 94°C for 35 min and stored at -80°C until hybridizations. Gene Chip hybridizations Whole genome microarrays (Human Genome U-133 Plus 2.0 arrays; Affymetrix) arrays, comprising 54,675 probe sets to analyze more than 47,000 transcripts including 38,500 well-characterized human genes, were used. Hybridizations, probe array scanning and gene expression analysis were performed at the Gene Chip Core Facility, Columbia University Genome Center. Each sample was hybridized once and each person contributed with 2 to 4 (median 3) tissue samples.