title: Development of software and modification of Q-FISH protocol for estimation of individual telomere length in immunopathology.
abstract: Telomere length is an important indicator of proliferative cell history and potential. Decreasing telomere length in the cells of an immune system can indicate immune aging in immune-mediated and chronic inflammatory diseases. Quantitative fluorescent in situ hybridization (Q-FISH) of a labeled (C3TA[Formula: see text] peptide nucleic acid probe onto fixed metaphase cells followed by digital image microscopy allows the evaluation of telomere length in the arms of individual chromosomes. Computer-assisted analysis of microscopic images can provide quantitative information on the number of telomeric repeats in individual telomeres. We developed new software to estimate telomere length. The MeTeLen software contains new options that can be used to solve some Q-FISH and microscopy problems, including correction of irregular light effects and elimination of background fluorescence. The identification and description of chromosomes and chromosome regions are essential to the Q-FISH technique. To improve the quality of cytogenetic analysis after Q-FISH, we optimized the temperature and time of DNA-denaturation to get better DAPI-banding of metaphase chromosomes. MeTeLen was tested by comparing telomere length estimations for sister chromatids, background fluorescence estimations, and correction of nonuniform light effects. The application of the developed software for analysis of telomere length in patients with rheumatoid arthritis was demonstrated.
authors.affiliation: * Laboratory of the Clinical Immunopathology, Research Institute of Fundamental and Clinical Immunology, 630099, Yadrintsevskaya Street 14, Novosibirsk, Russia.
authors.forename: M Sh
authors.initials: MS
authors.lastname: Barkovskaya
authors.affiliation: * Laboratory of the Clinical Immunopathology, Research Institute of Fundamental and Clinical Immunology, 630099, Yadrintsevskaya Street 14, Novosibirsk, Russia.|‡ Novosibirsk State University, 630090, Pirogova Street 2, Novosibirsk, Russia.
authors.forename: A G
authors.initials: AG
authors.lastname: Bogomolov
authors.affiliation: * Laboratory of the Clinical Immunopathology, Research Institute of Fundamental and Clinical Immunology, 630099, Yadrintsevskaya Street 14, Novosibirsk, Russia.
authors.forename: N Yu
authors.initials: NY
authors.lastname: Knauer
authors.affiliation: † Laboratory of the Morphology and Function of Subcellular Components, Institute of Cytology and Genetics SB RAS, 630090, Prospekt Lavrentyeva 10, Novosibirsk, Russia.|‡ Novosibirsk State University, 630090, Pirogova Street 2, Novosibirsk, Russia.
authors.forename: N B
authors.initials: NB
authors.lastname: Rubtsov
authors.affiliation: * Laboratory of the Clinical Immunopathology, Research Institute of Fundamental and Clinical Immunology, 630099, Yadrintsevskaya Street 14, Novosibirsk, Russia.
authors.forename: V A
authors.initials: VA
authors.lastname: Kozlov
mesh_terms: D001172:Arthritis, Rheumatoid / Q000235:genetics*; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D012984:Software; D016615:Telomere; D059505:Telomere Homeostasis
keywords: Quantitative fluorescence in situ hybridization (Q-FISH); image analysis; immunopathology; integrated fluorescence intensity (IFI); peptide nucleic acid (PNA); telomere length