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.     

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.     

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.     

利用流式荧光原位杂交法测定细胞端粒长度

目的建立利用流式荧光原位杂交法检测细胞端粒长度的技术方法。方法以端粒酶敲除的G3小鼠和同龄野生型小鼠为检测对象,分离其外周血中的单个核细胞后与肽核酸荧光探针杂交,用流式细胞仪采集和分析其端粒长度,分别用荧光原位杂交方法和SYBR Green荧光定量PCR方法验证其准确性。结果流式荧光原位杂交法测定G3小鼠细胞端粒相对长度与C57BJ/6野生型小鼠相比为0.5345,荧光定量PCR测定端粒相对长度为0.5717,结果基本一致。结论流式细胞术与原位杂交方法结合起来检测细胞端粒的平均长度可靠易行,对单个核细胞端粒平均长度的检测有较高的实用性。     

The controversial telomeres of lily plants

The molecular structure of the exceptional telomeres of six plant species belonging to the order Asparagales and two species of the order Liliales was analyzed using Southern blot and fluorescence in situ hybridization. Three different situations were found, namely: i) In the two Liliales species, Tulipa australis (Liliaceae) and Merendera montana (Colchicaceae), the chromosome ends display hybridization signals with oligonucleotides resembling telomere repeats of both plants (TTTAGGG)n and vertebrates (TTAGGG)n. ii) Asparagales species such as Phormium tenax (Hemerocallidaceae), Muscari comosum (Hyacinthaceae), Narcissus jonquilla (Amaryllidaceae) and Allium sativum (Alliaceae) lack both the plant telomere repeats and the vertebrate telomere repeats. iii) Two other Asparagales species, Aloe vera (Asphodelaceae) and an Iris hybrid (Iridaceae), display positive hybridization with the vertebrate telomere repeats but not with the plant telomere repeats. Southern blot hybridization revealed concurring results. On this basis, the composition of the telomere structure in this plant group is discussed.     

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 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.     

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

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.     

Conservation of (TTAGG)(n) telomeric sequences among ants (Hymenoptera,Formicidae)

To determine the telomere sequence in Tapinoma nigerrimum, we carried out in situ hybridization using TTAGGG and TTAGG repeat polymerase chain reaction (PCR)-generated probes. No hybridization signals were found when TTAGGG was used as a probe. However, strong signals were observed at the end of the chromosomes with the TTAGG probe. Southern blot analysis carried out on genomic DNA using TTAGG as a probe showed a strong hybridization signal even under highly stringent conditions. Similar results were obtained in Southern blot analysis carried out on genomic DNA of 19 species of ants belonging to three different subfamilies. In accordance with all the results shown in this article, the TTAGG repeat seems to be the major component of the telomere sequence in the majority of ant species.     

The telomere lengthening conundrum—artifact or biology?

Recent longitudinal studies of age-dependent leukocyte telomere length (LTL) attrition have reported that variable proportions of individuals experience LTL lengthening. Often, LTL lengthening has been taken at face value, and authors have speculated about the biological causation of this finding. Based on empirical data and theoretical considerations, we show that regardless of the method used to measure telomere length (Southern blot or quantitative polymerase chain reaction-based methods), measurement error of telomere length and duration of follow-up explain almost entirely the absence of age-dependent LTL attrition in longitudinal studies. We find that LTL lengthening is far less frequent in studies with long follow-up periods and those that used a high-precision Southern blot method (as compared with quantitative polymerase chain reaction determination, which is associated with larger laboratory error). We conclude that the LTL lengthening observed in longitudinal studies is predominantly, if not entirely, an artifact of measurement error, which is exacerbated by short follow-up periods. We offer specific suggestions for design of longitudinal studies of LTL attrition to diminish this artifact.     

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.     

Acquisition of telomere repeat sequences by transfected DNA integrated at the site of a chromosome break.

Previous analysis of plasmid DNA transfected into 108 cell clones demonstrated extensive polymorphism near the integration site in one clone. This polymorphism was apparent by Southern blot analysis as diffuse bands that extended over 30 kb. In the present study, nucleotide sequence analysis of cloned DNA from the integration site revealed telomere repeat sequences at the ends of the integrated plasmid DNA. The telomere repeat sequences at one end were located at the junction between the plasmid and cell DNA. The telomere repeat sequences at the other end were located in the opposite orientation in the polymorphic region and were shown by digestion with BAL 31 to be at the end of the chromosome. Telomere repeat sequences were not found at this location in the plasmid or parent cell DNA. Although the repeat sequences may have been acquired by recombination, a more likely explanation is that they were added to the ends of the plasmid by telomerase before integration. Comparison of the cell DNA before and after integration revealed that a chromosome break had occurred at the integration site, which was shown by fluorescent in situ hybridization to be located near the telomere of chromosome 13. These results demonstrate that chromosome breakage and rearrangement can result in interstitial telomere repeat sequences within the human genome. These sequences could promote genomic instability, because short repeat sequences can be recombinational hotspots. The results also show that DNA rearrangements involving telomere repeat sequences can be associated with chromosome breaks. The introduction of telomere repeat sequences at spontaneous or ionizing radiation-induced DNA strand breaks may therefore also be a mechanism of chromosome fragmentation.     

Impartial comparative analysis of measurement of leukocyte telomere length/DNA content by Southern blots and qPCR

Telomere length/DNA content has been measured in epidemiological/clinical settings with the goal of testing a host of hypotheses related to the biology of human aging, but often the conclusions of these studies have been inconsistent. These inconsistencies may stem from various reasons, including the use of different telomere length measurement techniques. Here, we report the first impartial evaluation of measurements of leukocyte telomere length by Southern blot of the terminal restriction fragments and quantitative PCR (qPCR) of telomere DNA content, expressed as the ratio of telomeric product (T)/single copy gene (S) product. Blind measurements on the same samples from 50 donors were performed in two independent laboratories on two different occasions. Both the qPCR and Southern blots displayed highly reproducible results as shown by r values > 0.9 for the correlations between results obtained by either method on two occasions. The inter-assay CV measurement for the qPCR was 6.45%, while that of the Southern blots was 1.74%. The relation between the results generated by Southern blots versus those generated by qPCR deviated from linearity. We discuss the ramifications of these findings with regard to measurements of telomere length/DNA content in epidemiological/clinical circumstances.     

Automatic telomere length measurements in interphase nuclei by IQ-FISH.

To benefit from the fluorescence-based automatic microscope (FLAME), we have adapted a PNA FISH technique to automatically determine telomere length in interphase nuclei. The method relies on the simultaneous acquisition of pan-telomeric signals and reference probe signals. We compared the quantitative figures to those for existing methods, i.e. Southern blot analysis and quantitative FISH (Q-FISH). Quantitative-FISH on interphase nuclei (IQ-FISH) allows the exact quantification of telomere length in interphase nuclei. Thus, this enables us to obtain not only exact information on the telomere length, but also morphological and topological details. The automatic measurement of large cell numbers allows the measurement of statistically relevant cell populations.     

Association of telomere instability with senescence of porcine cells

Telomeres are essential for the maintenance of genomic stability, and telomere dysfunction leads to cellular senescence, carcinogenesis, aging, and age-related diseases in humans. Pigs have become increasingly important large animal models for preclinical tests and study of human diseases, and also may provide xeno-transplantation sources. Thus far, Southern blot analysis has been used to estimate average telomere lengths in pigs. Telomere quantitative fluorescence in situ hybridization (Q-FISH), however, can reveal status of individual telomeres in fewer cells, in addition to quantifying relative telomere lengths, and has been commonly used for study of telomere function of mouse and human cells. We attempted to investigate telomere characteristics of porcine cells using telomere Q-FISH method. The average telomere lengths in porcine cells measured by Q-FISH correlated with those of quantitative real-time PCR method (qPCR) or telomere restriction fragments (TRFs) by Southern blot analysis. Unexpectedly, we found that porcine cells exhibited high incidence of telomere doublets revealed by Q-FISH method, coincided with increased frequency of cellular senescence. Also, telomeres shortened during subculture of various porcine primary cell types. Interestingly, the high frequency of porcine telomere doublets and telomere loss was associated with telomere dysfunction-induced foci (TIFs). The incidence of TIFs, telomere doublets and telomere loss increased with telomere shortening and cellular senescence during subculture. Q-FISH method using telomere PNA probe is particularly useful for characterization of porcine telomeres. Porcine cells exhibit high frequency of telomere instability and are susceptible to telomere damage and replicative senescence.     

Telomere length measurement in mouse chromosomes by a modified Q-FISH method

Telomeres are physical ends of mammalian chromosomes that dynamically change during the lifetime of a cell or organism. In order to understand mechanisms responsible for telomere dynamics, it is necessary to develop methods for accurate telomere length measurement. The most sensitive method for measuring telomere length in mouse chromosomes is quantitative fluorescence in situ hybridization (Q-FISH). The usual protocol for Q-FISH requires plasmids with variable numbers of telomeric repeats and fluorescence beads as calibration standards. Here, we describe a Q-FISH protocol in which two mouse lymphoma cell lines with well-defined telomere lengths are used as calibration standards. Using this protocol we demonstrate that reproducible results can be obtained in a set of four different mouse cell lines. This method can be adapted so that any pair of mammalian cell lines can serve as an internal calibration standard.     

Real-time quantitative PCR assay for measurement of avian telomeres

We present the application of a real-time quantitative PCR assay, previously developed to measure relative telomere length in humans and mice, to two bird species, the zebra finch Taeniopygia guttata and the Alpine swift Apus melba . This technique is based on the PCR amplification of telomeric (TTAGGG)_n sequences using specific oligonucleotide primers. Relative telomere length is expressed as the ratio (T/S) of telomere repeat copy number (T) to control single gene copy number (S). This method is particularly useful for comparisons of individuals within species, or where the same individuals are followed longitudinally. We used glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a single control gene. In both species, we validated our PCR measurements of relative telomere length against absolute measurements of telomere length determined by the conventional method of quantifying telomere terminal restriction fragment (TRF) lengths using both the traditional Southern blot analysis (Alpine swifts) and in gel hybridization (zebra finches). As found in humans and mice, telomere lengths in the same sample measured by TRF and PCR were well correlated in both the Alpine swift and the zebra finch.. Hence, this PCR assay for measurement of bird telomeres, which is fast and requires only small amounts of genomic DNA, should open new avenues in the study of environmental factors influencing variation in telomere length, and how this variation translates into variation in cellular and whole organism senescence.     

Simultaneous flow cytometric analysis of two cell surface markers,telomere length,and DNA content

BACKGROUND: Various protocols for estimation of telomere length in individual cells by flow cytometry using fluorescence in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes (Flow-FISH) have been described. Combined analysis of telomere length and cell phenotype, however, remains difficult because few fluorochromes with suitable emission spectra tolerate the harsh conditions needed for DNA denaturation during hybridization of the telomere-specific PNA probe. We overcame these problems and developed a method for measuring telomere length in cell subsets characterized by the expression of two surface antigens. METHODS: Alexa Fluor 488 and Alexa Fluor 546 were used for cell surface staining. Antigen-antibody complexes were covalently cross-linked onto the cell membrane before Flow-FISH. Cells were hybridized with a PNA probe conjugated to cyanine 5 (Cy5). Hoechst 33342 (HO342) was added for determination of cellular DNA content. For assay standardization, we added an aliquot of a single batch of 1,301 cells to each sample as an internal control before hybridization with the PNA probe. Samples were prepared in duplicate and analyzed on a standard three-laser BD LSR flow cytometer. For assay validation, the same samples were analyzed in parallel to correlate the percentage of telomere length of the sample versus 1,301 control cells to the mean size of terminal restriction fragments (TRFs) of DNA as determined by Southern gel analysis. RESULTS: The method permitted clear identification of lymphocyte subsets in samples hybridized for Flow-FISH, with subset frequencies comparable to those of untreated samples. At a concentration of 10 nM, the Cy5-labeled telomere-specific PNA probe produced a bright fluorescence signal well separated from background. Addition of HO342 in low concentration did not interfere with Cy5 telomere fluorescence, produced adequate DNA histograms, and permitted clear identification of cell phenotype. The probe concentration of 10 nM also proved optimal for inclusion of 1,301 control cells for assay standardization. Telomere length estimations by the current method correlated highly with TRF calculations by Southern gel hybridization (r(2)= 0.9, P = 0.0003). Application of our protocol to the analysis of human CD8CD28 lymphocyte subsets showed that CD8(+bright)CD28(-) lymphocytes generally exhibit shorter telomeres than CD8(+bright)CD28(+) cells. These data concurred with previous results of telomere shortening in CD8(+)CD28(-) T cells that were obtained by using different techniques. CONCLUSIONS: The multiparameter Flow-FISH protocol permitted rapid determination of differences in telomere length in subpopulations characterized by two surface markers without prior cell separation.     

In aged primary T cells,mitochondrial stress contributes to telomere attrition measured by a novel imaging flow cytometry assay

The decline of the immune system with age known as immune senescence contributes to inefficient pathogen clearance and is a key risk factor for many aged‐related diseases. However, reversing or halting immune aging requires more knowledge about the cell biology of senescence in immune cells. Telomere shortening, low autophagy and mitochondrial dysfunction have been shown to underpin cell senescence. While autophagy has been found to control mitochondrial damage, no link has been made to telomere attrition. In contrast, mitochondrial stress can contribute to telomere attrition and vice versa. Whereas this link has been investigated in fibroblasts or cell lines, it is unclear whether this link exists in primary cells such as human lymphocytes and whether autophagy contributes to it. As traditional methods for measuring telomere length are low throughput or unsuitable for the analysis of cell subtypes within a mixed population of primary cells, we have developed a novel sensitive flow‐FISH assay using the imaging flow cytometer. Using this assay, we show a correlation between age and increased mitochondrial reactive oxygen species in CD8⁺ T‐cell subsets, but not with autophagy. Telomere shortening within the CD8⁺ subset could be prevented in vitro by treatment with a ROS scavenger. Our novel assay is a sensitive assay to measure relative telomere length in primary cells and has revealed ROS as a contributing factor to the decline in telomere length.