title: Real-time quantitative polymerase chain reaction analysis of patients with refractory chronic periodontitis.
abstract: BACKGROUND
  Periodontitis is a complex multifactorial disease and is typically polygenic in origin. Genes play a fundamental part in each biologic process forming complex networks of interactions. However, only some genes have a high number of interactions with other genes in the network and may, therefore, be considered to play an important role. In a preliminary bioinformatic analysis, five genes that showed a higher number of interactions were identified and termed leader genes. In the present study, we use real-time quantitative polymerase chain reaction (PCR) technology to evaluate the expression levels of leader genes in the leukocytes of 10 patients with refractory chronic periodontitis and compare the expression levels with those of the same genes in 24 healthy patients.
  
  
  METHODS
  Blood was collected from 24 healthy human subjects and 10 patients with refractory chronic periodontitis and placed into heparinized blood collection tubes by personnel trained in phlebotomy using a sterile technique. Blood leukocyte cells were immediately lysed by using a kit for total RNA purification from human whole blood. Complementary DNA (cDNA) synthesis was obtained from total RNA and then real-time quantitative PCR was performed. PCR efficiencies were calculated with a relative standard curve derived from a five cDNA dilution series in triplicate that gave regression coefficients >0.98 and efficiencies >96%. The standard curves were obtained using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and growth factor receptor binding protein 2 (GRB2), casitas B-lineage lymphoma (CBL), nuclear factor-KB1 (NFKB1), and REL-A (gene for transcription factor p65) gene primers and amplified with 1.6, 8, 40, 200, and 1,000 ng/μL total cDNA. Curves obtained for each sample showed a linear relationship between RNA concentrations and the cycle threshold value of real-time quantitative PCR for all genes. Data were expressed as mean ± SE (SEM). The groups were compared to the analysis of variance. A probability value <0.01 was considered statistically significant.
  
  
  RESULTS
  The present study agrees with the preliminary bioinformatics analysis. In our experiments, the association of pathology with the genes was statistically significant for GRB2 and CBL (P <0.01), and it was not statistically significant for REL-A and NFKB1.
  
  
  CONCLUSIONS
  This article lends support to our preliminary hypothesis that assigned an important role in refractory aggressive periodontitis to leader genes.
authors.affiliation: Nanoworld Institute, Inter-University Center for Research and Teaching activity on Organic and Biological Nanoscience and Nanotechnology, University of Genoa, Italy.
authors.forename: Simone
authors.initials: S
authors.lastname: Marconcini
authors.forename: Ugo
authors.initials: U
authors.lastname: Covani
authors.forename: Antonio
authors.initials: A
authors.lastname: Barone
authors.forename: Orazio
authors.initials: O
authors.lastname: Vittorio
authors.forename: Michele
authors.initials: M
authors.lastname: Curcio
authors.forename: Serena
authors.initials: S
authors.lastname: Barbuti
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Scatena
authors.forename: Lamberto
authors.initials: L
authors.lastname: Felli
authors.forename: Claudio
authors.initials: C
authors.lastname: Nicolini
mesh_terms: D000328:Adult; D010520:Aggressive Periodontitis / Q000235:genetics*; D055113:Chronic Periodontitis / Q000235:genetics*; D019295:Computational Biology; D018450:Disease Progression; D005260:Female; D051380:GRB2 Adaptor Protein / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D020022:Genetic Predisposition to Disease / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D008297:Male; D008875:Middle Aged; D052002:NF-kappa B p50 Subunit / Q000235:genetics; D050722:Oncogene Protein v-cbl / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D051996:Transcription Factor RelA / Q000235:genetics
chemical_list: D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D052002:NF-kappa B p50 Subunit; C489548:NFKB1 protein, human; D050722:Oncogene Protein v-cbl; D051996:Transcription Factor RelA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases