Telomere length measurement-caveats and a critical assessment of the available technologies and tools

Abstract

Studies of telomeres and telomere biology often critically rely on the detection of telomeric DNA and measurements of the length of telomere repeats in either single cells or populations of cells. Several methods are available that provide this type of information and it is often not clear what method is most appropriate to address a specific research question. The major variables that need to be considered are the material that is or can be made available and the accuracy of measurements that is required. The goal of this review is to provide a comprehensive summary of the most commonly used methods and discuss the advantages and disadvantages of each. Methods that start with genomic DNA include telomere restriction fragment (TRF) length analysis, PCR amplification of telomere repeats relative to a single copy gene by Q-PCR or MMQPCR and single telomere length analysis (STELA), a PCR-based approach that accurately measures the full spectrum of telomere lengths from individual chromosomes. A different set of methods relies on fluorescent in situ hybridization (FISH) to detect telomere repeats in individual cells or chromosomes. By including essential calibration steps and appropriate controls these methods can be used to measure telomere repeat length or content in chromosomes and cells. Such methods include quantitative FISH (Q-FISH) and flow FISH which are based on digital microscopy and flow cytometry, respectively. Here the basic principles of various telomere length measurement methods are described and their strengths and weaknesses are highlighted. Some recent developments in telomere length analysis are also discussed. The information in this review should facilitate the selection of the most suitable method to address specific research question about telomeres in either model organisms or human subjects.

Figure 1. Extra-telomeric regions detected using different telomere measurement techniques

Telomere length measured by TRF includes subtelomeric DNA (green bar) and telomere variant repeats (red bars) which can occupy up to 2 kb of the start of the telomere. This “X” region overestimates the length of pure telomeric repeats (blue bars), and is variable based on the choice and location of restriction enzymes (represented as scissors). Some selected restriction enzymes will cut a particular common variant repeat (red scissor), and restriction sites will differ between chromosomes and individuals. STELA encompasses the variant and pure telomere repeats, but since the primer site is known (green arrow), the subtelomeric length can be subtracted (white box). Both Q-FISH and Flow-FISH use probes that are specific for pure telomeric repeats, although it is unknown whether arrays of pure telomere repeats in the variant region affect measurements. Q-PCR compares amplification of pure telomere reads using telomeric primers (blue arrows) to a single copy locus (orange) amplified with a second primer set (orange arrows). N.B. Techniques calibrated to TRF results will include the “X” region in the calculated telomere length, even if “X” is not detected in the method itself.

Figure 2. Inter-assay variability in flow FISH (A and B)

Each flow FISH telomere length measurement includes duplicate assays of telomere-PNA probe stained versus unstained conditions (or 4 tubes per test). Average median telomere length fluorescence from duplicate measurements of the same blood sample in over 59 independent experiments are plotted; measurements for gated lymphocytes (L, blue) are shown (panel A). To control for differences in hybridization, each condition (tube) includes an internal hybridization control consisting of diploid primary cells with a stable karyotype, bovine thymocytes (T, green), which are identical between each experimental plate set up and are used to normalize the fluorescence data (panel B). The average median telomere length calculated for this sample over the 59 independent experiments is 6.8 kb with a standard deviation of 0.2kb (average CV=3.3%). Intra-assay variability on flow FISH (C and D). Comparison between ~1000 consecutive replicate telomere length measurement tests were compared for lymphocytes (C) and granulocytes (D). Regression analysis showed correlation between replicates was 0.991 and 0.968 for lymphocytes and granulocytes respectively. Outlier sample replicates with CV>10%, 4/1052 (0.38%) for lymphocytes (red), and 18/995 (1.8%) for granulocytes (black) were excluded from analysis.