Robust Measurement of Telomere Length in Single Cells

Robust measurement of telomere length in single cells

Fang Wanga,b, Xinghua Panc,1, Keri Kalmbacha, Michelle L. Seth-Smitha, Xiaoying Yeb, Danielle M. F. Antumesa,d,e, Yu Yinb, Lin Liub,1, David L. Keefea,1, and Sherman M. Weissmanc,1

aDepartment of Obstetrics and Gynecology, New York University Langone Medical Center, NY 10016; bState Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071, China; cDepartment of Genetics, Yale University School of Medicine, New Haven, CT 06520-8005; dDepartment of Pathology, Fluminense Federal University, 24033-900, Rio de Janeiro, Brazil; and eCoordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) Foundation, Ministry of Education of Brazil, 70040-020, Brasilia, Brazil

Contributed by Sherman M. Weissman, April 9, 2013 (sent for review February 5, 2013) Measurement of telomere length currently requires a large popula- length for a given cell population (10–12). High-throughput tion of cells, which masks telomere length heterogeneity in single Q-FISH, flow FISH, and single telomere length analysis can be used cells, or requires FISH in metaphase arrested cells, posing technical for telomere measurement of dividing, nondividing, and senescent challenges. A practical method for measuring telomere length in cells, but these methods also require large cell populations (13–15). single cells has been lacking. We established a simple and robust The ability to measure telomere length in single cells rather approach for single-cell telomere length measurement (SCT-pqPCR). than relying upon average telomere length in cell populations or We first optimized a multiplex preamplification specific for telo- the entire tissue enables the study of biological heterogeneity on meres and reference genes from individual cells, such that the ampli- a cell-by-cell basis, an issue of fundamental importance for studies con provides a consistent ratio (T/R) of telomeres (T) to the reference of aging, development, carcinogenesis, and many other diseases. genes (R) by quantitative PCR (qPCR). The average T/R ratio of multi- Here, we demonstrate an accurate determination of telomere ple single cells corresponded closely to that of a given cell population length in individual cells, with the resolution and scalability of the measured by regular qPCR, and correlated with those of telomere qPCR telomere length assay. restriction fragments (TRF) and quantitative FISH measurements. Fur- The basis of qPCR is that within a given cell, the ratio of the copy thermore, SCT-pqPCR detected the telomere length for quiescent cells number of telomere repeats to the copy number of a multicopy that are inaccessible by quantitative FISH. The reliability of SCT-pqPCR fi fi reference gene is xed (3), and this method, because of its sim- also was con rmed using sister cells from two cell embryos. Telomere plicity, has been widely used to investigate a variety of telomere length heterogeneity was identified by SCT-pqPCR among cells of shortening-associated diseases (7), even sensitive enough to iden- various human and mouse cell types. We found that the T/R values tify mild telomere dysfunction resulting from chronological life of human fibroblasts at later passages and from old donors were stress (16, 17). We adapted qPCR to measure telomere length in lower and more heterogeneous than those of early passages and fi fi from young donors, that cancer cell lines show heterogeneous telo- individual cells by using a preampli cation step that speci cally mere lengths, that human oocytes and polar bodies have nearly iden- targets both the telomere and multicopy genes, followed by a qPCR tical telomere lengths, and that the telomere lengths progressively assay to obtain telomere to reference gene (T/R) ratio. A single-cell increase from the zygote, two-cell to four-cell embryo. This method telomere (SCT) length measurement method (SCT-pqPCR) runs will facilitate understanding of telomere heterogeneity and its role in robustly, and shows an identical T/R ratio for two sister blastomeres tumorigenesis, aging, and associated diseases. from two-cell–stage mouse embryos. The average result from SCT- qPCR with multiple single cells is linearly correlated to Q-FISH, single cell analysis | stem cell | cell senescence TRF, and conventional qPCR assays designed for a large number of cells. The heterogeneity of telomere length among several populations of cells by SCT-pqPCR run on multiple single cells is elomeres are the ribonucleoprotein structures that cap and — — Tprotect linear chromosome ends from genomic instability and consistent with and sometimes superior to results obtained by tumorigenesis (1, 2). Intriguingly, telomere shortening protects Q-FISH. Application of SCT-pqPCR to study telomere length against tumorigenesis by limiting cell growth (3, 4), but also can during early embryo development, aging, and cancer demonstrate impair tissue regenerative capability and cell viability (5, 6). the value of this single-cell telomere length assay method. Thus far, most assays of telomere length measure average telomere length from aggregates of many cells derived from Significance dissected tissues, cultured cells, or blood (7). Telomere restriction fragment (TRF) determination (1, 8), a Southern blot-based tech- Telomeres are the structures at the ends of chromosomes that nique, remains the “gold standard” for determining absolute telo- protect these ends from degradation or joining to one another. mere length, but requires a large amount of starting material (0.5–5 Telomeres consist of repeat DNA sequences and the length is μg DNA) and several days for processing. Moreover, the require- gradually eroded as the cell ages. The ability to measure telo- ments for gel electrophoresis and hybridization limit the scalability mere length in individual cells would be important for studies of this assay. Recently, a quantitative PCR (qPCR)-based method of cell senescence, malignancy, stem cell renewal, and human for telomere length measurement was developed, providing the fertility. We have developed a robust and practical method for convenience and scalability of PCR (9). Although the DNA re- estimating the telomere length of single cells, and used this quirement (35 ng) for qPCR is significantly less than TRF, it still method to demonstrate the heterogeneity or changes of relies on populations of cells to derive sufficient amount of DNA. telomere length in several systems. Quantitative FISH (Q-FISH) allows sensitive visualization of relative telomere length from individual cells and individual telo- Author contributions: X.P., L.L., D.L.K., and S.M.W. designed research; F.W. and K.K. meres, but this method requires many cells or metaphase arrested performed research; M.L.S.-S., X.Y., and Y.Y. contributed new reagents/analytic tools; F.W., X.P., D.M.F.A., L.L., D.L.K., and S.M.W. analyzed data; and F.W., X.P., K.K., L.L., cells, which precludes its application to many sample types, in- D.L.K., and S.M.W. wrote the paper. cluding postmitotic cells, senescent cells, and other nondividing The authors declare no conflict of interest. cells, and when only one actual cell is required to test. In addition, 1To whom correspondence may be addressed. E-mail: [email protected], liutelom@ preparing chromosome spreads requires significant technical skill, yahoo.com, [email protected], or [email protected]. and only proliferating cells within a population reach metaphase This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. stage, so this analysis potentially biases the estimates of telomere 1073/pnas.1306639110/-/DCSupplemental.

E1906–E1912 | PNAS | Published online May 9, 2013 www.pnas.org/cgi/doi/10.1073/pnas.1306639110 Downloaded by guest on October 1, 2021 Results genes, some with thousands of copies of sequences throughput the PNAS PLUS Establishment of Single-Cell qPCR for Telomere Length Measurement. genome, similar to telomeres, could provide more dependable am- Asinglecellpossesses∼6–7 pg of genomic DNA (18), but the plification. We tested three multicopy genes (Alu, B1,and18S rRNA) original qPCR assay requires nanogram amounts of DNA to as reference genes and found that the relative telomere lengths measure telomere length (9). To analyze telomere length in single measured with T/R for populations of human cells using multicopy cells, we developed a strategy based on qPCR of the telomere genes Alu or 18S rRNA by qPCR were consistent with the results versus reference gene after parallel preamplification of these tar- using the single-copy gene (36B4)(Fig. S1A). The correlation be- get sequences. The preamplification step consists of a multiplex tween repeat Alu and 36B4 (R2 = 0.9836) was slightly better than PCR for a limited number of cycles in a single tube containing between 18S rRNA and 36B4 (R2 = 0.9527) (Fig. 1B). a single cell without physical gDNA extraction, to generate suffi- We also measured the absolute telomere length of human cient DNA for downstream qPCR, yet retain the natural ratio of cell lines with the TRF method (Fig. S1B). The telomere length the targeted sequences (Fig. 1A). of human cell lines by TRF was proportional to the T/R ratio, Robust and faithful preamplification of the telomere se- and again the multicopy gene Alu (R2 = 0.8963) better corre- quence and reference genes from single cells, without distor- lated with TRF than did 18S rRNA (R2 = 0.7064) (Fig. 1C). The tion of their native ratio, is critical for this assay. To avoid slight distortion of 18S rRNA may aise from an outlier in the DNA loss during physical purification, we used a lysis buffer measurement. We chose the multicopy gene Alu for human cells containing EDTA and Nonidet P-40 to gently release DNA, or the B1 sequence for mouse cell as our reference gene in retain DNA integrity, and remain compatible with the sub- single-cell telomere analysis, and suggest taking 18S rRNA as an sequent PCR. In addition, we tested various reference genes, alternative when required. from a single-copy gene to several multicopy genes, to nor- With single-cell DNA or amounts up to 10 ng DNA from HeLa S3 malize the telomere units. The single-copy gene, 36B4, serves as cells, the PCR reached a plateau when the cycle number was more a reference gene in the conventional qPCR for telomere mea- than 20, regardless of the primers used. The mouse tail-tip fibroblast surement (9). However, 36B4 did not produce robust results for (TTF) showed a similar result (Fig. S2B). We preamplified single- single-cell amplification, and the melting curve of 36B4 ampli- cell DNA from human lung fibroblasts using telomere and Alu pri- cons (mouse and human) frequently showed multiple peaks, in mers simultaneously for 20, 18, 16, 14, or 12 cycles and found the Ct contrast to very robust telomere DNA amplification (see for exam- value proportionally increased with decreasing cycle number from ple, Fig. S2A), suggesting that the single-copy gene from a single cell 18 to 14 (Fig. S1E). To determine the optimal pre-PCR cycle may be unavailable for amplification in many conditions. Multicopy number, we prepared standard curves for telomere and reference GENETICS

Fig. 1. Design of SCT-pqPCR. (A) Schematic diagram of SCT-pqPCR. Green and red dots represent telomere sequence and reference gene (Alu) sequence in the chromosome, respectively. Green and red lines (thin) represent PCR productions by Tel and Alu primers, respectively, after preampliﬁcation. (B) Linear correlation analysis of relative telomere length of a population of cells by regular qPCR between the single-copy gene and multicopy genes as the reference gene. (C) Linear correlation analysis of telomere length measurement of a population of cells by TRF and regular qPCR with multicopy genes.

Wang et al. PNAS | Published online May 9, 2013 | E1907 Downloaded by guest on October 1, 2021 genes by amplification of a series of dilutions of genomic DNA. The single-cell telomere length by SCT-pqPCR significantly correlated standard curves were quite linear within DNA concentrations from with average telomere length of cell populations measured by 1ng/μLto16ng/μL for human cells (Fig. S2 C and D)and0.375ng/ conventional qPCR and Q-FISH; the Pearson test produced P μLto6ng/μL for mouse cells (Fig. S2C). A comparison of the Ct values of 0.001 and 0.006, respectively (Fig. 2 A and B). We also value of standard DNA (Fig. S2D) and the Ct value after different analyzed the correlation between SCT-pqPCR and TRF (Fig. numbers of pre-PCR cycles (Fig. S2E) indicated that pre-PCR cycle S1B) in human cell lines (F171, F200, HeLa S3, RuES2, and numbers from 16 to 18 were optimal; therefore, we chose the pre- SaOS2). The Pearson test again showed that the average of single- amplification cycle number 17 for human cells and 16 for mouse cells, cell telomere lengths measured by SCT-pqPCR was highly corre- because the telomere length of mouse generally is longer than that of lated with absolute telomere length by TRF, with a P value of 0.015 C human and, expectedly, requires fewer cycles for amplification. We (Fig. 2 ). Interestingly, we found lower correlation between fi D should point out that the T/R ratio obtained for mouse telomeres Q-FISH and TRF in these ve cell lines (Fig. 2 ). Presumably, cannot be directly compared with T/R ratio for human telomeres. We the heterogeneity of single-cell telomere lengths in cancer cell suggest that a correlation of T/R ratio measured by SCT-pqPCR with lines exceeds that of normal cell lines. In addition, variations TRF (kb) by Southern blot should be established initially using a cell in telomere lengths between dividing and nondividing cells may population for each species and for each laboratory. bias Q-FISH results, because Q-FISH measures telomere lengths only of dividing cells capable of arrest at metaphase, but the TRF Correlation of Average Telomere Length Estimated by SCT-pqPCR and and qPCR methods measure average telomere length of all cells. fi Telomere Length of Cell Populations by Conventional Methods. Al- This nding further supports the value of the SCT-pqPCR method though the results above demonstrated that the telomere length for telomere measurement of any individual cells independent of of single cells can be measured by our assay, it was necessary to their replication rate or potential. To further validate our assay, we measured telomere lengths in compare average telomere length calculated by averaging the fi results of single-cell measurements by SCT-pqPCR to established dilutions of preampli ed DNA of F171 and F200 after pre- amplifying DNA with target primers (telomere and reference methods, including qPCR, Q-FISH, and TRF, which measure gene Alu). When the input DNA was 2 ng, the relative telomere average telomere length of cell populations. Based on the above length in F171 was longer than that of F200 (Fig. S4), but when data, we compared telomere length of a population of cells mea- the DNA quantity decreased to 0.4 ng, there was no significant sured by conventional qPCR (Fig. S1A) and Q-FISH (Fig. 3 and difference in telomere lengths between fibroblast F171 and Fig. S3) to average telomere length of single cells measured by F200, P > 0.05 (Fig. S4). Therefore, when the prepurified DNA SCT-pqPCR in human cell lines F171, F200, HeLa S3, RuES2, drops below threshold value, one aliquot of the diluted DNA U2OS, and SaOS2 (Table S1). As expected, the data showed that does not fairly represent the entire genome. The approximately 0.5-ng to 1-ng threshold for purified human genome DNA was observed in a whole genome amplification effort (19, 20). The locus representation was significantly distorted when the input gDNA aliquoted from a large DNA pool is <0.5–1 ng. On the other hand, an intact single cell, although it contains only about 6–7pgDNA, contains an entire set of genomic sequences including all telomeres.

Validation of Single-Cell Telomere Length Measurements by SCT- pqPCR Using Various Assays. To validate single-cell telomere length measurements using our method, we chose two human cell types with different telomere lengths: HeLa S3 and 1301 human cell lines with average telomere lengths of 5 kb (15) and 70 kb, respectively. We also studied two mouse cell lines with different telomere lengths: embryonic stem cell (ESC) and TTF (21). The telomere length for each single cell in the same population varied by SCT-pqPCR analysis, and these results were consistent with the Q-FISH telomere lengths (Fig. 3 A, C, D,andF). Not unexpectedly, the calculated average telomere length of multiple single cells was significantly longer in mESCs than TTF in mice, and longer in 1301 human cells than HeLa S3 by SCT-pqPCR. The single-cell telomere length variations were found by SCT-pqPCR, and the variation also was identified by Q-FISH, with an independent sampled t test. The average T/R ratio of single cells measured by SCT- pqPCR was consistent with that of a cell population measured by SCT-pqPCR (T/R) or by conventional qPCR (T/S) (S, single-copy B E Fig. 2. Linear correlation of relative telomere length between single cells gene) (Fig. 3 and ). and their populations with various human cell lines. (A) Average telomere The limitation for the validation step above was that SCT-pqPCR length in single cells as T/R ratio by SCT-pqPCR is significantly correlated with and Q-FISH could not be performed for the same individual cells, that of a cell population shown as the T/S ratio by regular qPCR. The Pearson and no somatic cells with identical telomere lengths could be reliably test is applied. (B) Average telomere length in single cells by SCT-pqPCR is identified for this validation. To further validate the SCT-pqPCR significantly correlated with quantitative telomere length by Q-FISH. (C)Av- assay, we measured telomere length in pairs of sister cells derived erage telomere length of single human cells by SCT-pqPCR is highly correlated from two-cell mouse embryos and oocytes. Two-cell mouse embryos with absolute telomere length of population cells by TRF. (D) Quantitative exhibit identical telomere lengths between sister blastomeres (22), telomere length of metaphase cells by Q-FISH is correlated with the absolute and human polar bodies (PBs) show telomere lengths nearly iden- telomere length of cell populations by TRF. The trapezoid represents HeLa S3 cancer cell, the hexagon represents human lung fibroblast from a 71-y-old tical to their matched MII oocytes by Q-FISH analysis (23). Telo- donor (F200), the pentagon represents human lung fibroblast from a 14-wk mere lengths were remarkably similar between sister blastomeres by gestation (F171), the triangle represents SaOS2 cells, the square represents SCT-pqPCR, P >> 0.1 (Fig. 4A and Table S2), although the one-way human ESC (RuES2), and the circle represent U2OS cell. ANOVA (Tukey test) indicated that differences existed between

E1908 | www.pnas.org/cgi/doi/10.1073/pnas.1306639110 Wang et al. Downloaded by guest on October 1, 2021 PNAS PLUS

Fig. 3. Measurement of relative telomere length by SCT-pqPCR. (A and D) Analysis of single-cell telomere length by SCT-pqPCR (A and D) and QFISH (A′ and D′) in mouse (TTF and mES) and human cell lines (HeLa S3 and 1301). T/R ratio is against the multicopy gene B1 and Alu for mouse and human cells, respectively. The cycle number of pre-PCR is 16 and 17 for mouse and human cells, respectively. n = 6. Fluorescence intensity represents the telomere length signal by the Q-FISH method. (B and E) Validation of average telomere length of single cells by comparing to the average telomere length of cell populations in mouse and human cells. (B and E) Average telomere length of single cells as mean of T/R ratio by SCT-pqPCR. n =10.(B′ and E′) Average telomere length of cell populations as T/S ratio by regular qPCR. n = 6. Bar is ± SD. (C and F) Telomere length distribution in metaphase chromosomes of mouse and human cell populations by Q-FISH. Average telomere length as telomere ﬂuorescence unit (TFU) is indicated as mean ± SD.

pairs of sister cells from different embryos. Correlation analysis F204 P14 (from a 35-y-old donor) was longer (P < 0.05) than that GENETICS showed the telomere lengths between sister cells were proportional of human fibroblast F200 P7 (from a 71-y-old donor) by Q-FISH by the Pearson test (P =0.005)(Fig.4B). Telomere lengths between and conventional qPCR, but the average telomere length did not human oocytes (O) and their PBs demonstrated no significant dif- differ (P > 0.05) between F171 P16 and F204 P14 (Fig. S5). We ferences by the paired samples t test, t = 0.603, P =0.569(Fig.4C then analyzed the single-cell telomere lengths between F171 P16 and Table S2). Occasional (e.g., PB1/O1, PB6/O6) PB and oocytes and F200 P7 by SCT-pqPCR. Remarkably, the telomere lengths exhibited different telomere lengths, which could represent de- of single cells differed in the same population of both F171 P16 generation in telomere DNA or biological differences. Curiously and F200 P7 cells. Indeed, some single cells from F200 P7 had telomere lengths of oocytes and PBs in patients 6 and 7 were re- longer telomeres than F171 P16, as measured by SCT-pqPCR, markably longer than other patients by one-way ANOVA test, P < a finding confirmed by Q-FISH analysis (Fig. 5 A and B and Fig. 0.001 (Fig. 4C). The telomere lengths of human oocytes correlated S3). The coefficient of variation (CV) showed single-cell telo- highly with the paired PB telomere lengths by liner correlation mere length in F200 P7 to be more heterogeneous than F171 P16 analysis (Fig. 4D). Measurements of telomere length in pairs of (Table 1). When human fibroblasts were continuously cultured human oocytes and PBs by SCT-pqPCR agreed with our previous (F171 P16 to P31 and F200 from P7 to P12), metaphase chro- studies (23). Unlike sister cells of mouse two-cell embryos, telomere mosome spreads were rarely available for analysis of cells at later lengths between daughter cells of HeLa S3 frequently showed dif- passage, presumably because these cells had undergone senes- ferences, although the correlation between telomere lengths in cence and failed to divide. Interestingly, SCT-pqPCR demon- daughter cells was still high (Fig. 4 E and F,andTable S2). strated increased variation in telomere length among cells at later passage compared with early passages (CV 0.486 in F171 SCT-pqPCR Identifies Telomere Length and Its Heterogeneity in Single P31 and 0.398 in F200 P12 vs. 0.169 in F171 P16 and 0.233 in Cells from Various Cell Types. We used SCT-pqPCR to measure F200 P7, respectively) (Fig. 5A and Table 1). telomeres of human lung fibroblast cell lines from different aged The above data show that telomere length varies among in- donors and passage numbers. When comparing telomere length dividual cells in a given population. We also analyzed telomere in cell populations, the average telomere length of human fi- length of single cells in human ESC cultures (RuES2, telomerase broblast F171 passage number (P)16 (from 14-wk gestation) and activity-positive), cultured cancer cells (HeLa S3, telomerase

Wang et al. PNAS | Published online May 9, 2013 | E1909 Downloaded by guest on October 1, 2021 Fig. 4. Measurements of telomere length in mouse two-cell stage embryos and pairs of human oocytes and PBs by SCT-pqPCR. (A) Measurement of single cell telomere length by SCT-pqPCR between two sister cells from a mouse two-cell (2c) embryo. n =6.(B) Telomere length is highly correlated between two sister cells of a two-cell embryo. (C) Comparison of telomere length in pairs of human MII oocytes and PBs by SCT-pqPCR. n =6.(D) The telomere length of human oocyte is proportional to that of the paired PB. (E) Measurement of single-cell telomere length by SCT-pqPCR between two daughter cells from the HeLa S3. H represents HeLa S3. n = 6. Bar is ± SD. (F) The telomere length correlation of two daughter cells for 10 pairs of cells of HeLa S3.

activity-positive), and cultured osteosarcoma cells (U2OS and ated by TRF, Q-FISH, and conventional qPCR. SCT-pqPCR shows SaOS2, telomerase activity-negative) by SCT-pqPCR. As expec- that the average telomere length and its variation within multiple ted, average telomere length and variability among cells of a given single cells for human lung fibroblasts, embryos, and cancer cells, population differed among these various cell types (Fig. 5A,and correlate with data obtained by conventional qPCR and Q-FISH Fig. S3 A and C). The telomere lengths of HeLa S3 cells varied with the corresponding populations of cells. Using this method, we more than other cell types, with a CV of 0.294 (Table 1). Telo- found that the T/R value of early passage human fibroblasts to be mere lengths of single cells at metaphase in these cells (HeLa S3, relatively uniform, but later passages of human fibroblasts and Rues2, SaOS2, and U2OS) also showed extensive variations when cancer cells become increasingly variable, that human oocytes and measured by Q-FISH, confirming that single-cell telomere length PBs share nearly identical telomere length (T/R), and that the varies within the same cell population (Fig. 5B,andFig. S3 B and telomere length (T/R) is significantly increased from zygote to two- D). From the CV of different human cell lines as estimated by cell and to four-cell embryos. SCT-pqPCR not only allows mea- SCT-pqPCR and Q-FISH, the single-cell telomere lengths in surement of telomere length in single cells, independent of their cancer cell populations vary more than in early passage human ability to divide, but also provides a tool to estimate the hetero- fibroblast cell lines (Table 1). Similarly, fibroblasts from older geneity of telomere length in cell populations. people show more heterogeneity in telomere length than those One key to the successful establishment of our assay was iden- of young people, and cells at later passages show telomere tifying reliable multicopy genes, Alu and B1, to normalize telomere length variations more than those at earlier passages. measurements. It is possible that aneuploidy may exist in cancer In addition, as a very delicate case, we also successfully mea- cells and embryos, and single loci may drop out during lysis and sured the telomere lengths of individual cells from mouse zygote pre-PCR. The use of a multicopy gene also may help avoid am- to four-cell embryos (Fig. 5C), and the average telomere length plification bias from single cells because it targets an abundant increased along with development of mouse embryos by SCT- sequence, similar to the abundant telomere units, within the single pqPCR (Fig. 5D). Consistently, telomere length in developing cells. Because of the large number of Alu (500,000 copies) or B1 mouse embryos was found to lengthen significantly from zygotes to (150,000 members per genome) family units and their interspersion the four-cell embryo stage (22). throughout the genome, small copy number variations, such as a deletion of a few megabases or even a few hundred new insertions, Discussion would not substantially affect the denominator. When desired, two Here we report the development and validation of single-cell reference genes [i.e., Alu (or B1)and18S rRNA] may be used in telomere assay by qPCR (SCT-pqPCR), which provides sim- combination in one assay. If the copy number of the reference gene ple, robust and high-quality analysis of telomere length in in- (Alu or B1) were to change extremely significant among the test dividual cells. SCT-pqPCR can measure the telomere length samples, 18S rRNA may be used alone to replace Alu (or B1)asthe with an actual single cell, for example, each sister cell of a two- reference gene. Another key is the single-tube preamplification of cell mouse embryo, which are confirmed identical in T/R ratio. A the reference gene in parallel with the telomere sequences for linear relationship was demonstrated between average T/R values a limited number of cycles by multiplex PCR, which avoids sample generated with SCT-pqPCR and absolute telomere length gener- loss, and retains a faithful ratio of products, yet significantly expands

E1910 | www.pnas.org/cgi/doi/10.1073/pnas.1306639110 Wang et al. Downloaded by guest on October 1, 2021 methylation, and other functional genomics technologies. The PNAS PLUS significance of measuring telomere length in individual cells is ev- ident in several respects. This process will facilitate the identifica- tion and functional studies of cancer stem cells and pluripotent stem cells, and distinguish them from other cells in mixed populations (24), and advance our understanding of the molecular mechanisms underlying variation of individual cells in development, disease, senescence, and tumorigenesis (25). Single-cell telomere estimation also may be particularly useful to predict the viability and chromosomal stability of oocytes and embryos for women undergoing assisted reproductive technologies. Methods Single-Cell Isolation and DNA Extraction. Human and mouse cell lines were cultured in regular media. Mouse embryos and human germinal vesicle oocytes were cultured in K -modified simplex optimized medium and human tubal fluid, respectively. The single cells were isolated and lysed in a PCR tube without physical purification before the first PCR. Genomic DNA of cell populations was extracted by the tissue and blood DNA extraction kit (Qiagen) following the manufacturer’s instructions. The details are shown in SI Methods.SeeFig. S6 for derivation of daughter cells and Table S3 for the sequence of primers used by qPCR. Fig. 5. Variations of telomere length in single cells. (A and B) A summary of telomere length for multiple individual cells measured by SCT-pqPCR (A) and Single-Cell Genomic DNA Amplification by PCR (pre-PCR) with Primers of Q-FISH (B), shown as box-plot. The small circle represents an outlier; the Telomere and Reference Gene, and Product Purification. Pre-PCR was per- asterisk represents extreme value. The result for each individual cell is shown formed using DNA Polymerase Hot Start Version (TAKARA). The reactions in Fig. S3. F171 and F200 are human lung fibroblast from a 14-wk gestation were set up by aliquoting 38 μL of master mix into the 0.2 mL PCR tubes each and a 71-y-old donor, respectively. P16, P31, P7, and P12 refer to passage with 2 μL single-cell genomic DNA. Each reaction was set up with by 4 μL10× number. HeLa S3: human cancer cell. RuES2: human embryonic stem cell. PCR buffer, 4 μL 2.5 mM dNTP, 0.25 μL DNA polymerase, 1 μL each of telo- U2OS and SaOS2: human osteosarcoma. Single-cell telomere length of hu- μ μ man fibroblasts shortened with the age and passage number, and the het- mere forward and reverse primer (10 M), and 1 L each of multicopy gene μ μ erogeneity of F171 and F200 increased with the passage. The later passage forward and reverse primer (10 M) or single gene primer 36B4 (10 M) and μ fi F171 P31 and F200 P12 were unavailable for metaphase telomere Q-FISH enough water to make up a 40- L nal volume. Thermal cycler reaction because of cell senescence and compromised division. (C) Telomere length- conditions were set at 94 °C for 5 min followed by different cycles of 94 °C fi ening of single cells derived from mouse zygotes to four-cell embryos by for 15 s, 60 °C annealing for 30 s and extension at 72 °C for 30 s, with a nal fi SCT-pqPCR. Z, zygote; 2c, two-cell embryo; 4c, four-cell embryo. n =6.(D) extension for 10 min at 72 °C. PCR products were puri ed following the fi Analysis of average telomere length in different stage mouse embryos by protocol of the puri cation Kit (DNA clean and concentrator-5; Zymo Re- fi μ Student Newman–Keuls test shows that telomere length elongates from search). Finally the puri ed PCR products were eluted in 64 L of double zygote to four-cell embryo. n = 4. Bar is ± SD. distilled water.

Q-PCR Assay for Average Telomere Measurement of a Cell Population. Average material for measurements by qPCR. We found that SaOS2 cells telomere length was measured from total genomic DNA of human cell lines proliferated more slowly. In addition, many cells undergo senes- by using the qPCR method previously described (9). First we followed the same protocol and chose the single-copy gene (36B4) as the reference. In cence and fail to reach metaphase stage, and thus cannot be mea- addition, we chose multicopy genes (Alu and 18S rRNA) as reference genes sured by Q-FISH. These data demonstrate that SCT-pqPCR has an and measured the average telomere length of different human cell lines. GENETICS advantage over Q-FISH for telomere length at single-cell levels Each reaction included 10 μL2× SYBR Green mix (Bio-Rad), 0.5 μL each of because it is not dependent on cell division and does not bias results 10 μM forward and reverse primers, 4 μL molecular-filter water and 5 μL when testing a heterogeneous population of cells consisting of both genomic DNA (7 ng/μL) to yield a 20-μL reaction. DNA samples were placed dividing and quiescent cells. in adjacent three wells of a 96-well plate for telomere primers and ref- SCT-pqPCR is a simple, efficient, and accurate method for erence gene primers, respectively. A Bio-Rad thermocycler (CFX system test) was used with reaction conditions of 95 °C for 10 min followed by 40 measuring cell-to-cell differences and heterogeneity in telomere cycles of data collection at 95 °C for 15 s, 60 °C anneal for 30 s and 72 °C length at the molecular level, comparable to RNA-seq, CpG extend for 30 s along with 80 cycles of melting curve from 60 °C to 95 °C. To serve as a reference for standard curve calculation, mouse TTF and human fibroblast were serially diluted and qPCR performed as described above. Table 1. Variation of single-cell telomere length by SCT-pqPCR After thermal cycling was completed, the CFX manager software was used and Q-FISH in different cell lines to generate standard curves and Ct values for telomere signals and reference gene signals. Here we used different reference genes for telomere length Q-FISH (fluorescence measurement, and each sample of DNA had one telomere signal and ref- T/R ratio intensity) erence gene signal. The average telomere length was termed the T/R ratio instead of the T/S ratio. Cell line Mean SD CV Mean SD CV Q-PCR Assay for Single-Cell Telomere Measurement. Telomere length of single F171 P16 0.845 0.143 0.169 422.652 77.626 0.184 cells was measured by qPCR after pre-PCR, purification and aliquoting. The ——— F171 P31 0.749 0.364 0.486 reaction was set up with SYBR Green I in 96-wells plates. The purified F200 P7 0.710 0.165 0.233 249.855 67.251 0.269 products of Pre-PCR for each single cell were aliquoted with 5 μL into each F200 P12 0.724 0.289 0.398 ———well of a 96-well plate. Three repeat reactions were performed for each RuES2 1.274 0.299 0.234 578.253 138.486 0.239 sample plus each pair of primers. The final master mix of each well in the HeLa S3 0.680 0.200 0.294 339.530 113.267 0.334 qPCR was 10 μL2× SYBR Green mix, 0.5 μL each of forward and reverse SaOS2 1.202 0.291 0.242 625.677 240.469 0.384 primer (10 μMforTel,Alu, B1,and36B4)and4μL of molecular-filtered U2OS 1.872 0.419 0.224 1550.925 468.434 0.302 water. The qPCR conditions were the same as above. The results were analysis by CFX manager software and the relative telomere length of single The cycle number of pre-PCR is 17. n =6. cells was calculated by the T/R ratio.

Wang et al. PNAS | Published online May 9, 2013 | E1911 Downloaded by guest on October 1, 2021 Telomere Q-FISH. Telomere Q-FISH and quantiﬁcation were performed as lyzed with one-way ANOVA. The frequency of telomere distribution was described previously (11, 22). compared by a nonparametric test. The correlation analysis was performed by the Pearson correlation test; α is set at 0.05 for all tests. Southern Blot Analysis of TRF. Telomere restriction fragment analysis was performed as described by using the TeloTAGGG Telomere Length Assay Kit ACKNOWLEDGMENTS. We thank Susan Smith (New York University Langone (Roche) according to the protocol provided by the manufacturer. The mean Medical Center) for providing the U2OS and Hela S3 cancer cell line, and telomere length was calculated using the following (26): TRF = ∑(ODi)/ Brigitte L. Arduini (Laboratory of Molecular Vertebrate Embryology, Human ∑(ODi/Li), where ODi is the chemiluminescent signal and ODi/Li is the length Pluripotent Stem Cell Core Facility (The Rockefeller University) for providing of the TRF at position. the human embryonic stem cell RuES2. This work was supported by funding from the New York University Department of Obstetrics and Gynecology and the Clinical and Translational Science Institute at New York University Data Statistics. All of the data statistics were obtained by SPSS 13.0 software. (NIH1UL1RR029893); the Most National Major Basic Research Program The P value for comparison of two groups was derived from the in- (2009CB941000, 2011CBA01002); and National Institute of Health Grants dependent-samples t test. The multiple groups’ statistical data were ana- 1P01GM099130-01 and 1R21HD066457-01.

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