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Forensic Application of Telomere Shortening in Age-at-Death Estimation
Published in Sara C. Zapico, Mechanisms Linking Aging, Diseases and Biological Age Estimation, 2017
Quantitative-Fluorescence In Situ Hybridization (Q-FISH): uses directly fluorescently labeled (CCCTAA) 3 Peptide Nucleic Acid (PNA) probes as a high-affinity alternative to DNA oligonucleotide probes that specifically hybridize to denature telomere DNA repeat arrays. Then, the fluorescent signal can be detected and measured relative to standards of known telomere length in metaphase spreads with specific software for Q-FISH image analysis (Poon and Lansdorp 2001). Q-FISH is the method of choice for high-resolution telomere length measurements at specific chromosome ends. Q-FISH has also been used to detect ends without detectable repeats (0.5 kb) as well as chromosome fusion events.
Deducing the cellular mechanisms associated with the potential genotoxic impact of gold and silver engineered nanoparticles upon different lung epithelial cell lines in vitro
Published in Nanotoxicology, 2022
Samantha V. Llewellyn, Wolfgang J. Parak, Jonas Hühn, Michael J. Burgum, Stephen J. Evans, Katherine E. Chapman, Gareth J. S. Jenkins, Shareen H. Doak, Martin J. D. Clift
For telomeric visualization, Q-FISH was performed whilst Flow-FISH was adopted for telomere length quantification. Both A549 and 16HBE14o− were cultured for 72-h post-seeding at 37 °C and 5% CO2 before a 24-h Au (8.68 × 10−3 mg/mL) and Ag (1.66 × 10−5 mg/mL) ENP exposure was performed. Following ENP exposure, the supernatants were harvested and replaced with 0.1 μg/mL of KaryoMAXTM ColcemidTM (Gibco®, UK) diluted in cell culture medium and incubated for 4-h at 37 °C and 5% CO2. Cells were then trypsinized, centrifuged, and washed twice in PBS. Centrifugation was repeated once more and the cell pellet was re-suspended in hypotonic solution (55 mM KCl and 20 mM Hepes, pH 7.4) for 10 min at RT to induce cell swelling. After which, a fixative solution (3:1, methanol to glacial acetic acid) was added and the samples mixed gently. Samples were centrifuged and re-suspended in fresh fixative solution twice, before being stored at 4 °C for up to 72-h prior to in situ hybridization as described in Figure 1.
The effect of maternal body mass index (BMI) and telomere function on in vitro fertilization (IVF) outcome: a preliminary cohort study
Published in Human Fertility, 2020
Natalie Weeg, Anat Hershko Klement, Einat Haikin, Aliza Amiel, Adrian Shulman, Tal Biron-Shental, Amir Wiser
FISH was performed using the probes hTERC, 13q and 15q and washed with 4′-6-diamidino-w-phenylindole (DAPI) to evaluate the number of telomerase copies and telomere capture; and Q-FISH with peptic nucleic acid (PNA) probe was undertaken to determine telomere length and aggregate formation. Senescence can be assessed through the microscope using either FISH or Q-FISH. Both FISH and Q-FISH were based on a similar standard laboratory protocol, as detailed below.
Telomeres as a molecular marker of male infertility
Published in Human Fertility, 2019
Ewa Boniewska-Bernacka, Anna Pańczyszyn, Natalia Cybulska
Three research methods are most commonly used to determine telomere length in spermatozoa (Table 1). These are: (i) Southern blot (TRF: terminal restriction fragment); (ii) Q-FISH (quantitative fluorescence in situ hybridization); and (iii) real-time PCR.