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

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.     

FISH studies reveal the molecular and chromosomal organization of individual telomere domains in tomato

The molecular and cytological organization of the telomeric repeat (TR) and the subtelomeric repeat (TGR1) of tomato were investigated by fluorescence in situ hybridization (FISH) techniques. Hybridization signals on extended DNA fibres, visualized as linear fluorescent arrays representing individual telomeres, unequivocally demonstrated the molecular co-linear arrangement of both repeats. The majority of the telomeres consisted of a TR and a TGR1 region separated by a spacer. Microscopic measurements of the TR and TGR1 signals revealed high variation in length of both repeats, with maximum sizes of 223 and 1330 kb, respectively. A total of 27 different combinations of TR and TGR1 was detected, suggesting that all chromosome ends have their own unique telomere organization. The fluorescent tracks on the extended DNA fibres were subdivided into four classes: (i) TR–spacer–TGR1; (ii) TR–TGR1; (iii) only TR; (iv) only TGR1. FISH to pachytene chromosomes enabled some of the TR/TGR1 groups to be assigned to specific chromosome ends and to interstitial regions. These signals also provided evidence for a reversed order of the TR and TGR1 sites at the native chromosome ends, suggesting a backfolding telomere structure with the TGR1 repeats occupying the most terminal position of the chromosomes. The FISH signals on diakinesis chromosomes revealed that distal euchromatin areas and flanking telomeric heterochromatin remained highly decondensed around the chiasmata in the euchromatic chromosome areas. The rationale for the occurrence and distribution of the TR and TGR1 repeats on the tomato chromosomes are discussed.     

Measurement of telomere length in haematopoietic cells using in situ hybridization techniques

The DNA of human chromosomes terminates in several kilobases of telomere repeats that are gradually lost with; age and with replication in vitro. Defective telomere maintenance has been shown to be causally linked to cell cycle exit and apoptosis. In order to overcome the limitations imposed by Southern blotting, we have established a quantitative fluorescence in situ hybridization (Q-FISH) technique. This technique allows estimation of telomere length in specific chromosome arms from metaphase cell preparations. Furthermore, we have extended quantitative in situ hybridization to flow cytometry (flow FISH) in order to obtain information on the mean telomere repeat content in suspended cells. Telomere length in granulocytes, monocytes, CD8 and CD4 T lymphocytes and natural killer cells was found to differ slightly in the peripheral blood of adults. However, strikingly longer telomeres were observed in B lymphocytes (approximately 1.3 kb longer), suggesting a functional role for telomere maintenance in this cell subset. In summary, Q-FISH and flow FISH represent new methods for measuring telomere length in single cells and allow studies of telomere dynamics in haematopoietic subpopulations at various stages of normal and abnormal antigen responses.     

Validation and development of quantitative flow cytometry-based fluorescence in situ hybridization for intercenter comparison of telomere length measurement

BACKGROUND: Telomeres are highly conserved repeats at the ends of chromosomes that maintain chromosome stability and reflect the replicative potential of cells. Telomere length can be determined by Southern blot hybridization or quantitative fluorescence in situ hybridization (Q-FISH). Recently, two flow cytometry-based (Flow) FISH protocols have been published. METHODS: We compared the telomere length measured by Southern blotting and Flow FISH using standard beads to calibrate and quantify the fluorescence intensity. RESULTS: The telomeric fluorescence of cord blood and peripheral blood mononuclear cells was similar to that reported by other studies. There was a linear relationship between the telomeric fluorescence determined by Flow FISH and the telomere fragment size determined by Southern blotting (r = 0.89; P < 0.001). CONCLUSION: It is important to set up a center-specific curve and select appropriate cell lines for reference. This Q-Flow FISH protocol will facilitate the measurement of telomere length and allow more meaningful comparison of data (in standard fluorescence units or fragment size) between institutes.     

Telomere length measurement by fluorescence in situ hybridization and flow cytometry: tips and pitfalls

BACKGROUND: Telomeres containing noncoding DNA repeats at the end of the chromosomes are essential for chromosomal stability and are implicated in regulating the replication and senescence of cells. The gradual loss of telomere repeats in cells has been linked to aging and tumor development and methods to measure telomere length are of increasing interest. At least three methods for measuring the length of telomere repeats have been described: Southern blot analysis and quantitative fluorescence in situ hybridization using either digital fluorescence microscopy (Q-FISH) or flow cytometry (flow-FISH). Both Southern blot analysis and Q-FISH have specific limitations and are time-consuming, whereas the flow-FISH technique requires relatively few cells (10(5)) and can be completed in a single day. A further advantage of the flow-FISH method is that data on the telomere length from individual cells and subsets of cells (lymphocytes and granulocytes) can be acquired from the same sample. In order to obtain accurate and reproducible results using the flow-FISH technique, we systematically explored the influence of various steps in the protocol on telomere length values and established an acceptable range for the most critical parameters. METHODS: Isolated leukocytes from whole blood are denatured by heat and 70%/75% formamide, then hybridized with or without a telomere-specific fluorescein isothiocyante (FITC)-conjugated peptide nucleic acid probe (PNA). Unbound telomere PNA is washed away, the DNA is counterstained, and telomere fluorescence is measured on a flow cytometer using an argon ion laser (488 nm) to excite FITC. For each sample, duplicates of telomere PNA-stained and unstained tubes are analyzed. RESULTS: Cell counts and flow-FISH telomere length measurements were performed on leukocytes and thymocytes of humans and other species. Leukocyte suspensions were prepared by two red blood cell lysis steps with ammonium chloride. Optimal denaturation of DNA was achieved by heating at 85-87 degrees C for 15 min in a solution containing 70%/75% formamide. Hybridization was performed at room temperature with a 0.3 microg/ml telomere-PNA probe for at least 60-90 min. Unbound telomere-PNA probe was diluted at least 4,000-40,000 times with wash steps containing 70%/75% formamide at room temperature. LDS 751 and DAPI were suitable as DNA counterstains as they did not show significant interference with telomere length measurement. CONCLUSIONS: The use of flow-FISH for telomere length measurements in nucleated blood cells requires tight adherence to an optimized protocol. The method described here can be used to determine rapidly the telomere length in subsets of nucleated blood cells.     

LNA-modified oligonucleotides are highly efficient as FISH probes

Fluorescence in situ hybridization (FISH) is a highly useful technique with a wide range of applications including the delineation of complex karyotypes, prenatal diagnosis of aneuploidies, screening for diagnostic or prognostic markers in cancer cells, gene mapping and gene expression studies. However, it is still a fairly time-consuming method with limitations in both sensitivity and resolution. Locked Nucleic Acids (LNAs) constitute a novel class of RNA analogs that have an exceptionally high affinity towards complementary DNA and RNA. Substitution of DNA oligonucleotide probes with LNA has shown to significantly increase their thermal duplex stability as well as to improve the discrimination between perfectly matched and mismatched target nucleic acids. To exploit the improved hybridization properties of LNA oligonucleotides in FISH, we have designed several LNA substituted oligonucleotide probes specific to different human-specific repetitive elements, such as the classical satellite-2, telomere and alpha-satellite repeats. In the present study we show that LNA modified oligonucleotides are excellent probes in FISH, combining high binding affinity with short hybridization time.     

Telomere analysis by fluorescence in situ hybridization and flow cytometry.

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.     

Telomere shortening in Fanconi anaemia demonstrated by a direct FISH approach

Analysis of telomere status in patients with Fanconi anaemia (FA) has previously been carried out by measurement of telomere restriction fragment (TRF) length by Southern blotting and densitometry. Results from these studies indicated that FA patients had significant reduction in telomere length compared with age-matched controls. This paper confirms and extends these findings using a direct FISH technique, which showed that 15 out of 16 FA patients had increased loss of telomere signals compared with controls. In 12 out of the 16 patients, decrease in telomere signal intensity could also be detected using a Q-FISH approach.     

Visualization of the terminal structure of rice chromosomes 6 and 12 with multicolor FISH to chromosomes and extended DNA fibers

High-resolution fluorescence in situ hybridization (FISH) on interphase and pachytene nuclei, and extended DNA fibers enabled microscopic distinction of DNA sequences less than a few thousands of base pairs apart. We applied this technique to reveal the molecular organization of telomere ends in japonica rice (Oryza sativa ssp. japonica), which consist of the Arabidopsis type TTTAGGG heptameric repeats and the rice specific subtelomeric tandem repeat sequence A (TrsA). Southern hybridizations of DNA digested with Bal31 and EcoRI, and FISH on chromosomes and extended DNA fibers demonstrated that (1) all chromosome ends possess the telomere tandem repeat measuring 3–4 kb; (2) the subtelomeric TrsA occurs only at the ends of the long arms of chromosomes 6 and 12, and measure 6 and 10 kb, which corresponds to 231 and 682 copies for these sites, respectively; (3) the telomere and TrsA repeats are separated by at most a few thousands of intervening nucleotide sequences. The molecular organization for a general telomere organization in plant chromosomes is discussed.     

Telomere length measurements using digital fluorescence microscopy.

BACKGROUND: The ends of chromosomes (telomeres) are important to maintain chromosome stability, and the loss of telomere repeat sequences has been implicated in cellular senescence and genomic instability of cancer cells. The traditional method for measuring the length of telomeres (Southern analysis) requires a large number of cells (>10(5)) and does not provide information on the telomere length of individual chromosomes. Here, we describe a digital image microscopy system for measurements of the fluorescence intensity derived from telomere repeat sequences in metaphase cells following quantitative fluorescence in situ hybridization (Q-FISH). METHODS: Samples are prepared for microscopy using Q-FISH with Cy3 labeled peptide nucleic acid probes specific for (T(2)AG(3))(n) sequences and the DNA dye DAPI. Separate images of Cy3 and DAPI fluorescence are acquired and processed with a dedicated computer program (TFL-TELO). With the program, the integrated fluorescence intensity value for each telomere, which is proportional to the number of hybridized probes, is calculated and presented to the user. RESULTS: Indirect tests of our method were performed using simulated as well as defined tests objects. The precision and consistency of human telomere length measurements was then analyzed in a number of experiments. It was found that by averaging the results of less than 30 cells, a good indication of the telomere length (SD of 10-15%) can be obtained. CONCLUSIONS: We demonstrate that accurate and repeatable fluorescence intensity measurements can be made from Q-FISH images that provide information on the length of telomere repeats at individual chromosomes from limited number of cells.     

定量荧光原位杂交测定端粒长度

目的:应用定量荧光原位杂交(Q-FISH)方法测定端粒长度。方法:选取4种端粒长度均一的标准细胞株采用Q-FISH的方法做出荧光亮度与端粒长度的标准曲线,从而得出实验细胞株的端粒长度,与DNA印迹法测定末端限制性片段(TRF)长度进行二者之间的相关性分析。结果:检测荧光强度的最佳线性曝光时间为400ms,相对于DNA印迹法,定量荧光原位杂交(Q-FISH)法所需标本量少,实验周期短,端粒长度结果与Southern杂交法具有很好的相关性。结论:采用定量荧光原位杂交方法测端粒长度具有重复性好、精确可靠的特点,适用于对珍贵标本的端粒改变进行分析。     

Genetic dissection of the Kluyveromyces lactis telomere and evidence for telomere capping defects in TER1 mutants with long telomeres

In the yeast Kluyveromyces lactis, the telomeres are composed of perfect 25-bp repeats copied from a 30-nucleotide RNA template defined by 5-nucleotide terminal repeats. A genetic dissection of the K. lactis telomere was performed by using mutant telomerase RNA (TER1) alleles to incorporate mutated telomeric repeats. This analysis has shown that each telomeric repeat contains several functional regions, some of which may physically overlap. Mutations in the terminal repeats of the template RNA typically lead to telomere shortening, as do mutations in the right side of the Rap1p binding site. Mutations in the left half of the Rap1p binding site, however, lead to the immediate formation of long telomeres. When mutated, the region immediately 3' of the Rap1p binding site on the TG-rich strand of the telomere leads to telomeres that are initially short but eventually undergo extreme telomere elongation. Mutations between this region and the 3' terminal repeat cause elevated recombination despite the presence of telomeres of nearly wild-type length. Mutants with highly elongated telomeres were further characterized and exhibit signs of telomere capping defects, including elevated levels of subtelomeric recombination and the formation of extrachromosomal and single-stranded telomeric DNA. Lengthening caused by some Rap1 binding site mutations can be suppressed by high-copy-number RAP1. Mutated telomeric repeats from a delayed elongation mutant are shown to be defective at regulating telomere length in cells with wild-type telomerase, indicating that the telomeric repeats are defective at telomere length regulation.     

Assessment of telomere length and factors that contribute to its stability.

Short strands of tandem hexameric repeats known as telomeres cap the ends of linear chromosomes. These repeats protect chromosomes from degradation and prevent chromosomal end-joining, a phenomenon that could occur due to the end-replication problem. Telomeres are maintained by the activity of the enzyme telomerase. The total number of telomeric repeats at the terminal end of a chromosome determines the telomere length, which in addition to its importance in chromosomal stabilization is a useful indicator of telomerase activity in normal and malignant tissues. Telomere length stability is one of the important factors that contribute to the proliferative capacity of many cancer cell types; therefore, the detection and estimation of telomere length is extremely important. Until relatively recently, telomere lengths were analyzed primarily using the standard Southern blot technique. However, the complexities of this technique have led to the search for more simple and rapid detection methods. Improvements such as the use of fluorescent probes and the ability to sort cells have greatly enhanced the ease and sensitivity of telomere length measurements. Recent advances, and the limitations of these techniques are evaluated. Drugs that assist in telomere shortening may contribute to tumor regression. Therefore, factors that contribute to telomere stability may influence the efficiency of the drugs that have potential in cancer therapy. These factors in relation to telomere length are also examined in this analysis.     

Loss of chromosome 13 in cultured human vascular endothelial cells

In the vascular endothelium of human beings, telomere length is negatively related while the frequency of aneuploidy is positively related to donor age. Both in culture and in vivo the frequency of aneuploidy increases as telomere length is shortened. In this study we explored the relation between telomere length and aneuploidy in cultured human umbilical vein endothelial cells (HUVEC) by: (a) karyotype analysis and fluorescent in situ hybridization (FISH), (b) measurement of the terminal restriction fragments (TRF), and (c) assessment of replicative senescence by the expression of beta-galactosidase. Of 8 HUVEC strains, 7 cell strains lost chromosome 13, as shown by metaphase analysis and FISH of interphase cells. Five strains gained chromosome 11. In addition, five HUVEC strains became hypotetraploid shortly after the loss of chromosome 13. The loss of chromosome 13 was observed as early as PD 20, when mean TRF length was greater than 9 kb and the percentage of cells positive for beta-galactosidase was relatively low. The almost uniform loss of chromosome 13 suggests that this unique type of aneuploidy of HUVEC is the result of a progressive expression of clones with survival advantage.     

利用粗线期染色体和DNA纤维的FISH分析水稻端粒序列

利用粗线期染色体和DNA纤维的荧光原位杂交(FISH)技术分析了水稻广陆矮四号(Oryzasativassp.indicacv.GuangluaiNo.4)的端粒序列。粗线期染色体荧光原位杂交结果表明,大多数染色体的末端都有端粒串联重复,但信号的强度在不同染色体上是不同的。伸展DNA纤维荧光原位杂交结果显示,端粒最长的线状信号长度为6.55μm,最短的为1.82μm,依据2.51kb/μm的标准,它们分别相当于16.44kb和4.56kb。端粒的平均信号长度为3.62±1.32μm,相当于9.09±3.31kb。由此可以估计,最长的、最短的和平均长度的端粒拷贝数约为2349、651和1298±473。     

Tankyrase 2 poly(ADP-ribose) polymerase domain-deleted mice exhibit growth defects but have normal telomere length and capping

Regulation of telomere length maintenance and capping are a critical cell functions in both normal and tumor cells. Tankyrase 2 (Tnks2) is a poly(ADP-ribose) polymerase (PARP) that has been shown to modify itself and TRF1, a telomere-binding protein. We show here by overexpression studies that tankyrase 2, like its closely related homolog tankyrase 1, can function as a positive regulator of telomere length in human cells, dependent on its catalytic PARP activity. To study the role of Tnks2 in vivo, we generated mice with the Tnks2 PARP domain deleted. These mice are viable and fertile but display a growth retardation phenotype. Telomere analysis by quantitative fluorescence in situ hybridization (FISH), flow-FISH, and restriction fragment analysis showed no change in telomere length or telomere capping in these mice. To determine the requirement for Tnks2 in long-term maintenance of telomeres, we generated embryonic stem cells with the Tnks2 PARP domain deleted and observed no change, even upon prolonged growth, in telomere length or telomere capping. Together, these results suggest that Tnks2 has a role in normal growth and development but is not essential for telomere length maintenance or telomere capping in mice.     

Accelerated telomere shortening in the human inactive X chromosome

Telomeres are nucleoprotein complexes at the end of eukaryotic chromosomes, with important roles in the maintenance of genomic stability and in chromosome segregation. Normal somatic cells lose telomeric repeats with each cell division both in vivo and in vitro. To address a potential role of nuclear architecture and epigenetic factors in telomere-length dynamics, the length of the telomeres of the X chromosomes and the autosomes was measured in metaphases from blood lymphocytes of human females of various ages, by quantitative FISH with a peptide nucleic-acid telomeric probe in combination with an X-chromosome centromere-specific probe. The activation status of the X chromosomes was simultaneously visualized with antibodies against acetylated histone H4. We observed an accelerated shortening of telomeric repeats in the inactive X chromosome, which suggests that epigenetic factors modulate not only the length but also the rate of age-associated telomere shortening in human cells in vivo. This is the first evidence to show a differential rate of telomere shortening between and within homologous chromosomes in any species. Our results are also consistent with a causative role of telomere shortening in the well-documented X-chromosome aneuploidy in aging humans.     

In situ analysis of changes in telomere size during replicative aging and cell transformation

Telomeres have been shown to gradually shorten during replicative aging in human somatic cells by Southern analysis. This study examines telomere shortening at the single cell level by fluorescence in situ hybridization (FISH). FISH and confocal microscopy of interphase human diploid fibroblasts (HDFs) demonstrate that telomeres are distributed throughout the nucleus with an interchromosomal heterogeneity in size. Analysis of HDFs at increasing population doubling levels shows a gradual increase in spot size, intensity, and detectability of telomeric signal. FISH of metaphase chromosomes prepared from young and old HDFs shows a heterogeneity in detection frequency for telomeres on chromosomes 1, 9, 15, and Y. The interchromosomal distribution of detection frequencies was similar for cells at early and late passage. The telomeric detection frequency for metaphase chromosomes also decreased with age. These observations suggest that telomeres shorten at similar rates in normal human somatic cels. T-antigen transformed HDFs near crisis contained telomere signals that were low compared to nontransformed HDFs. A large intracellular heterogeneity in telomere lengths was detected in two telomerase-negative cell lines compared to normal somatic cells and the telomerase-positive 293 cell line. Many telomerase-negative immortal cells had telomeric signals stronger than those in young HDFs, suggesting a different mechanism for telomere length regulation in telomerase-negative immortal cells. These studies provide an in situ demonstration of interchromosomal heterogeneity in telomere lengths. Furthermore, FISH is a reliable and sensitive method for detecting changes in telomere size at the single cell level.     

Uncapping and deregulation of telomeres lead to detrimental cellular consequences in yeast

Telomeres are the protein-nucleic acid structures at the ends of eukaryote chromosomes. Tandem repeats of telomeric DNA are templated by the RNA component (TER1) of the ribonucleoprotein telomerase. These repeats are bound by telomere binding proteins, which are thought to interact with other factors to create a higher-order cap complex that stabilizes the chromosome end. In the budding yeast Kluyveromyces lactis, the incorporation of certain mutant DNA sequences into telomeres leads to uncapping of telomeres, manifested by dramatic telomere elongation and increased length heterogeneity (telomere deregulation). Here we show that telomere deregulation leads to enlarged, misshapen "monster" cells with increased DNA content and apparent defects in cell division. However, such deregulated telomeres became stabilized at their elongated lengths upon addition of only a few functionally wild-type telomeric repeats to their ends, after which the frequency of monster cells decreased to wild-type levels. These results provide evidence for the importance of the most terminal repeats at the telomere in maintaining the cap complex essential for normal telomere function. Analysis of uncapped and capped telomeres also show that it is the deregulation resulting from telomere uncapping, rather than excessive telomere length per se, that is associated with DNA aberrations and morphological defects.