It has long been presumed impossible to measure telomeres in vertebrate DNA by PCR amplification with oligonucleotide primers designed to hybridize to the TTAGGG and CCCTAA repeats, because only primer dimer-derived products are expected. Here we present a primer pair that eliminates this problem, allowing simple and rapid measurement of telomeres in a closed tube, fluorescence-based assay. This assay will facilitate investigations of the biology of telomeres and the roles they play in the molecular pathophysiology of diseases and aging.

Telomere measurement by quantitative PCR

https://www.cell.com/cell/pdf/S0092-8674(13)01602-4.pdf

Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System

Longevity, stress response, and cancer in aging telomerase-deficient mice.

Telomeres have emerged as crucial cellular elements in aging and various diseases including cancer. To measure the average length of telomere repeats in cells, we describe our protocols that use fluorescent in situ hybridization (FISH) with labeled peptide nucleic acid (PNA) probes specific for telomere repeats in combination with fluorescence measurements by flow cytometry (flow FISH). Flow FISH analysis can be performed using commercially available flow cytometers, and has the unique advantage over other methods for measuring telomere length of providing multi-parameter information on the length of telomere repeats in thousands of individual cells. The accuracy and reproducibility of the measurements is augmented by the automation of most pipetting (aspiration and dispensing) steps, and by including an internal standard (control cells) with a known telomere length in every tube. The basic protocol for the analysis of nucleated blood cells from 22 different individuals takes about 12 h spread over 2-3 days.

Flow cytometry and FISH to measure the average length of telomeres (flow FISH)

Telomeres and Telomerase

Determination of telomere length is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Fluorescence in situ hybridization (FISH) of telomere repeats has been used to calculate telomere length, a method called quantitative (Q)-FISH. We here present a quantitative flow cytometric approach, Q-FISHFCM, for evaluation of telomere length distribution in individual cells based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA)3probe and DNA staining with propidium iodide. A simple and rapid protocol with results within 30 h was developed giving high reproducibility. One important feature of the protocol was the use of an internal cell line control, giving an automatic compensation for potential differences in the hybridization steps. This protocol was tested successfully on cell lines and clinical samples from bone marrow, blood, lymph nodes and tonsils. A significant correlation was found between Southern blotting and Q-FISHFCMtelomere length values ( P = 0.002). The mean sub-telomeric DNA length of the tested cell lines and clinical samples was estimated to be 3.2 kbp. With the Q-FISHFCMmethod the fluorescence signal could be determined in different cell cycle phases, indicating that in human cells the vast majority of telomeric DNA is replicated early in S phase.

/pdf/telomere-analysis-by-fluorescence-in-situ-hybridization-and-3mxz04ea8w.pdf

Telomere analysis by fluorescence in situ hybridization and flow cytometry

Replication timing of human telomeric DNA and other repetitive sequences analyzed by fluorescence in situ hybridization and flow cytometry.

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Telomere analysis by fluorescence in situ hybridization and flow cytometry

Replication timing of human telomeric DNA and other repetitive sequences analyzed by fluorescence in situ hybridization and flow cytometry.