Here, we measure the telomere lengths of a variety of different species. Sirt1 contributes to telomere maintenance and augments. With tesla, but not trf analysis or qfish, we were able to quantitate the length of the shortest telomeres. Deletion of the telomerase reverse transcriptase gene and. Correlation between differentiation of adrenocortical. Guidelines for the selection of functional assays to. For analysis of chromosomal aberrations, metaphases were analyzed by superimposing the telomere image on the dapi image using the tfltelo software.
Telomere shortening rate predicts species life span pnas. Development of software and modification of qfish protocol for. Quantitative fluorescence in situ hybridization qfish request. The distribution pattern of critically short telomeres in.
To measure the average length of telomere repeats in. If there were fewer than 30 h and rs cells present on a diagnostic slide, it was insufficient for reliable statistical analysis, and the slide was not included. For tissue sections, the nuclei thus identified were manually selected by the operator as belonging to either epithelial or stromal groups. The automated image analysis was performed using a 3dmodelbased segmentation approach osterwald et al.
Threedimensional telomere dynamics in follicular thyroid. The captured images were analyzed using our own tissue analysis software, tissuetelo ver. Telomere length measurements using digital fluorescence. Each file of acquired images was saved in black and white 8bit and was loaded into the imagej software. Telomere shortening is associated with a number of common agerelated diseases. Pml induces compaction, trf2 depletion and dna damage. Cause and effect analysis was devised by professor kaoru ishikawa, a pioneer of quality management, in the 1960s. Metaphase chromosome preparation, qfish and image analysis. Image processing for telomere analysis is of increasing interest in many. Association of telomere instability with senescence of. The diagrams that you create with are known as ishikawa diagrams or fishbone diagrams because a completed diagram can look like the skeleton of a fish.
Qfish image analysis software is available for free from. We addressed this role directly in vivo by using a newly developed assay, which measures specifically the load of ultrashort single telomeres below. Samples are prepared for microscopy using qfish with cy3 labeled peptide nucleic acid probes speci. Obm genetics interphase quantitative fluorescence in. The fluorescent signal can then be detected and measured relative to standards of known telomere length in metaphase spreads with specific software for qfish image analysis for qfish freely available at. Using an array of unique proprietary technologies, life length addresses peoples aging process through telomere and telomerase measurements and diagnostics. Us national institute of health developed imagej software is described. The qfish method and software for qfish image analysis freely available from the flintbox network at has been, and continues to be, informative in the telomere. Highthroughput quantitative fluorescence in situ hybridization ht qfish is a highly accurate. Our methodology for qfish and image analysis for telomere measurement. Qfish can be applied for studying chromosome imbalances in interphase nuclei or. The qfish situ telomere length assay requires image analysis.
Image analysis software was a kind gift of peter s. Telomeres engaged in fusions and sister associations were excluded from qfish analysis. Computerassisted analysis of microscopic images can provide quantitative information on the. Fish signal areas were selected using rectangular selection tool to generate the plot of image containing the graph depicting the intensity profiles. All steps are performed using an established documented laboratory workflow, algorithms for image analysis, and quality control pedro et al. Fish is a powerful but technically challenging procedure. Quantitative image analysis was performed using the. Analytical validation of telomere analysis technology for the high. Ff and fm, fetal fibroblast and mesenchymal cells derived from fetus at embryonic day 50, respectively. A method for measuring the distribution of the shortest. Computerassisted analysis of microscopic images can provide quantitative information on the number of telomeric repeats in individual. The software calculates the overflow values and applies it to the data to obtain correct compensation data.
Software packages other than tfltelo may be available for qfish on a commercial basis. For image acquisition and analysis, sky requires specific hardware and software, comprising a customdesigned single triplebandpass filter and an interferometer able to retrieve spectral information for every pixel in a digital image. A ht qfish histograms of telomere length distributions of the indicated cell lines. Assessment of telomere length and factors that contribute to its stability. Image analysis was performed using a fujifilm bas 1500 bioimaging analysis system including image reader software version 1. Principles of qfish medical genetics central lakes medical. Splice variant analysis fish telomere length analysis qfish fish analysis of.
Journal of bioinformatics and computational biology vol. Telomeres have emerged as crucial cellular elements in aging and various diseases including cancer. For manual image acquisition, images were acquired with the leica tcs sp5 dmi6000 confocal laser scanning microscope using the las af software and parameters as described above. The exact causes of aging are still not understood, and it is unclear why some species live less than 1 d, while others can live more than 400 y. It requires cultured cells or fixed tissue sections. Qfish analysis on metaphase leukocytes, isolated from human. Qfish is carried out with a fluorescently labelled pna probe that binds with high specificity to telomeric sequences. Our highly scalable technologies provide a detailed look at ones cellular. Qfish image analysis software is available for free from the flintbox network at. Louis, mo for 3 hours and harvested for metaphase spreads. Telomerase gene therapy in adult and old mice delays aging.
Qfish technique required appropiate digital image analysis system designed to control acquisition of digital images, as well as to perform fluorescence intensity measurement 35. Digital ish image quantification and statistical analysis. American journal of respiratory and critical care medicine. Acapella high content imaging and analysis software. Assessment of telomere length and factors that contribute. All steps are performed using an established documented laboratory workflow, algorithms for image analysis, and quality control. Telomere length and structure analysis by telomere quantitative fluorescence in situ hybridization qfish.
Flow fish was performed as described elsewhere but with minor modi. Telomere fish analysis was performed using methods reported by osullivan 36. Telomere length assessment in tissue sections by quantitative fish. Threedimensional telomere signatures of hodgkin and reed. Trf analysis cells were included in agarose plugs, and trf analysis was performed as described previously blasco et al. The files generated by tfltelo can be exported to any standard software for statistical analysis. To ensure reproducibility of measurements by qfish, it is important to minimize sources of variation associated. The dna image plane was segmented using a watershed algorithm. Once the pnas specifically hybridize to denatured telomere dna repeats, the detected signal is compared to standards of known telomere length with specific qfish image analysis software. Telomere analysis using 3d fluorescence microscopy suggests. Tool for synteny analysis using mcscanx and visualization using circos evol2circos provide a tool based userfriendly platform to analyse user specific data for synteny construction and visualization, and a web based platform to facilitate the browsing of the syntenic information of different fish species using circos plot, bar, dual and dot plot. For karyotype analysis and quantitative analysis of telomeres, metaphase chromosomes were fixed and then hybridized using the peptide nucleic acidfish preparation method described previously poon and lansdorp, 2001a, poon and lansdorp, 2001b. Quantitative fluorescence in situ hybridization qfish springer. The fluorescent signal emitted by a telomere spot corresponds to its length and the integration of a dedicated image analysis software system permits the calculation of a combined fluorescent value from signals.
To determine whether telomere length in the alveolar cells in the emphysematous lungs was altered, we used computerassisted image analysis software to evaluate telomere signal intensity in type ii cells and endothelial cells in the lungs of the patients with emphysema, asymptomatic smokers, and nonsmokers. Quantitative fluorescence in situ hybridization qfish. The technique was then published in his 1990 book, introduction to quality control. To improve the quality of cytogenetic analysis after qfish, we. Measuring telomere length and telomere dynamics in.
A role of telomere shortening in osteoarthritis oa has been suggested, mainly based on the assessment of mean telomere length in ex vivo expanded chondrocytes. Correlation between telomere attrition of zona fasciculata. For southern blot analysis, 5 mg of genomic dna derived from the control and. The red color images of pi were converted to blue images for image analysis. Red rectangle, enlarged region shown in the right column. Quantitative fish image analysis for telomere length. The algorithm for each stage can be selected from a list of available algorithms, with selection available for subfunctions as well. The method allows the quantification of telomere length in distinct cell populations within a single sample by antibody staining. Quantitativefish analysis was performed using tfltelo image analysis software as described by poon et al. Investigation of telomere length dynamics in induced. Qfish is the method of choice for high resolution telomere length measurements at specific chromosome ends and for all chromosomes. After thawing, cells are assessed for viability, counted, seeded on 384well plates, and fixed. Telomere image analysis qfish images were captured by a ccd camera orcaer94, hamamatsu photonics kk, hamamatsu, japan mounted on an epifluorescence microscope 80i, nikon, tokyo, japan equipped with a triple bandpass filter set for 4.
The study reveals that telomere length is critical for meristem activity and stem cell renewal, thus coupling telomere dynamics to plant development. A cy3labeled ccctaa 3 peptide nucleic acid probe telo c fasmac, atsugi, catalogue. Quantitative fluorescent in situ hybridization qfish of a labeled c 3 ta 2 3 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. Life length is an industryleading biotechnology company working to improve peoples health worldwide. A representative images of telomere qfish in pig cells. Qfish has been used extensively to quantitate information regarding telomere length distribution and associating it with various illnesses. Highthroughput telomere length quantification by fish and. Nf and nm, newborn 7 or 8 days after birth fibroblast and. Quantitative fish qfish is a comprehensive method that allows. Flow cytometry and fish to measure the average length of.
Telomere length measurement caveats and a critical. Qfish analysis was performed on metaphase spreads that were collected and processed as described above from mefs or the bone marrow of mice femurs, and tfltelo image analysis software was used. After the images are prepared, the integrated fluorescence brightness is computed for each labelled structure of interest. Wildderived inbred mouse strains have short telomeres. Telomere fish image analyses were performed with optimas image analysis software media cybernetics, silver spring, md. Computerassisted analysis of microscopic images can provide quantitative. The fluorescent signal can then be detected and measured relative to standards of known telomere length in metaphase spreads with specific software for qfish image analysis freely available at. In this study, we have applied qfish to measure average telomere lengths in. Development of software and modification of qfish protocol. Pdf determination of telomere length by the quantitative. Measuring telomere length and telomere dynamics in evolutionary biology and ecology. It was developed by biomedical researchers in the early 1980s to detect and localize the presence or absence of specific dna sequences on chromosomes. Analyze using image capture software and telomere measurement tools.
If the fluorescence intensity reaches saturation, telomere lengths become underestimated. Fluorescence in situ hybridization fish is a molecular cytogenetic technique that uses fluorescent probes that bind to only those parts of a nucleic acid sequence with a high degree of sequence complementarity. In addition, we developed software to automatically quantify tesla results fig. Microscope control and image acquisition were performed using the image pro plus software package version 7.
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