Time: six weeks after commencement of periodontal therapy (after treatment end) Tissue: Peripheral blood monocytes Phenotype: severe periodontitis
Treatment protocol
Fifty ml of blood were obtained by venipucture and collected in Vacutainer cell preparation tubes (Beckton-Dickinson, NJ, USA) with sodium heparin, and were used to harvest peripheral blood mononuclear cells (PBMCs). A 1:1 dilution with PBS was made and the diluted blood sample was centrifuged at 1,000 g in Ficoll tubes (Sigma Accuspin, St. Louis, MO). After washing and counting of the cells in a phase hemacytometer (Hauser Scientific, Horsham, PA), PBMCs were re-suspended in 80 microlitres of MACS buffer (PBS pH 7.2, 0.5% BSA and 2 mM EDTA) and incubated on ice with 20 microlitres of CD14 microbeads (Miltenyi Biotec, Auburn CA) per 107 total cells for 15 minutes. After washings, the cell suspension was applied to a MACS LS separation column (Miltenyi Biotec) placed in the magnetic field of a MACS separator (Miltenyi Biotec). Thereafter, the column was removed from the separator and CD14 positive cells were collected by washings in MACS buffer.
Extracted molecule
total RNA
Extraction protocol
CD14 positive cells were vigorously pipetted in Trizol (Invitrogen Life Technologies, Carlsbad, CA, USA). After incubation with chloroform and centrifugation at 12,000 g, RNA in the upper aqueous phase was precipitated by mixing with isopropyl alcohol, further centrifugation and washing in 75% ethanol. Further purification of the extracted RNA was achieved by the use of the RNeasy total RNA isolation kit (Qiagen, Valencia, CA, USA) according to the manufacturer.s instructions. The purified RNA was quantified spectrophotometrically. One hundred nanograms of total RNA were amplified and reverse transcribed using the GeneChip expression 3.-amplification two-cycle cDNA synthesis kit (Affymetrix, Santa Clara, CA, USA) and the MEGAscript T7 transcription kit (Ambion, Austin, TX, USA) according to the manufacturers. instructions.
Label
biotin
Label protocol
Synthesis of biotin-labeled cRNA and subsequent fragmentation were performed by the use of the IVT labeling kit (Affymetrix, Santa Clara, CA, USA). The cRNA yield was determined spectrophotometrically at 260 nm. Fragmented cRNA was stored at -80C until hybridization.
Hybridization protocol
According to the manufacturer's instructions.
Scan protocol
According to the manufacturer's instructions.
Description
Patients were recruited among the ones seeking treatment at the clinic for post-doctoral Periodontics, Columbia University College of Dental Medicine. To be eligible for enrollment, patients had to be diagnosed with severe periodontitis, with radiographic evidence of bone loss extending to greater or equal to 30% of the tooth length at several teeth. Additional inclusion criteria required: age greater or equal to 18 years; presence of greater or equal to 2 teeth/quadrant with a pocket depth of greater or equal to 6 mm and concomitant attachment loss of greater or equal to 3 mm; a minimum of 20 teeth present; no prior periodontal therapy; no systemic conditions or genetic disorders that entail the diagnosis of periodontitis as a manifestation of systemic diseases; no use of systemic antibiotics or regular use of anti-inflammatory drugs for the preceding 6-month period; no diabetes mellitus; no current use of tobacco products or of nicotine replacement medication; not currently pregnant. A total of 15 subjects, 7 male and 8 female, were enrolled. All participants were informed about the scope and procedures of the study and informed consent was obtained.
Data processing
All CEL files in the series were processed using RMA in R using justRMA with default parameters.
