Robust measurement of telomere length in single cells

Establishment of Single-Cell qPCR for Telomere Length Measurement.

A single cell possesses ∼6–7 pg of genomic DNA (18), but the original qPCR assay requires nanogram amounts of DNA to measure telomere length (9). To analyze telomere length in single cells, we developed a strategy based on qPCR of the telomere versus reference gene after parallel preamplification of these target sequences. The preamplification step consists of a multiplex PCR for a limited number of cycles in a single tube containing a single cell without physical gDNA extraction, to generate sufficient DNA for downstream qPCR, yet retain the natural ratio of the targeted sequences (Fig. 1A).

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.

Robust and faithful preamplification of the telomere sequence and reference genes from single cells, without distortion of their native ratio, is critical for this assay. To avoid DNA loss during physical purification, we used a lysis buffer containing EDTA and Nonidet P-40 to gently release DNA, retain DNA integrity, and remain compatible with the subsequent PCR. In addition, we tested various reference genes, from a single-copy gene to several multicopy genes, to normalize the telomere units. The single-copy gene, 36B4, serves as a reference gene in the conventional qPCR for telomere measurement (9). However, 36B4 did not produce robust results for single-cell amplification, and the melting curve of 36B4 amplicons (mouse and human) frequently showed multiple peaks, in contrast to very robust telomere DNA amplification (see for example, Fig. S2A), suggesting that the single-copy gene from a single cell may be unavailable for amplification in many conditions. Multicopy genes, some with thousands of copies of sequences throughput the genome, similar to telomeres, could provide more dependable amplification. We tested three multicopy genes (Alu, B1, and 18S rRNA) as reference genes and found that the relative telomere lengths measured with T/R for populations of human cells using multicopy genes Alu or 18S rRNA by qPCR were consistent with the results using the single-copy gene (36B4) (Fig. S1A). The correlation between repeat Alu and 36B4 (R ^{= 0.9836) was slightly better than between} 18S rRNA and 36B4 (R ^{= 0.9527) (}Fig. 1B).

We also measured the absolute telomere length of human cell lines with the TRF method (Fig. S1B). The telomere length of human cell lines by TRF was proportional to the T/R ratio, and again the multicopy gene Alu (R ^{= 0.8963) better correlated with TRF than did} 18S rRNA (R ^{= 0.7064) (}Fig. 1C). The slight distortion of 18S rRNA may aise from an outlier in the measurement. We chose the multicopy gene Alu for human cells or the B1 sequence for mouse cell as our reference gene in single-cell telomere analysis, and suggest taking 18S rRNA as an alternative when required.

With single-cell DNA or amounts up to 10 ng DNA from HeLa S3 cells, the PCR reached a plateau when the cycle number was more than 20, regardless of the primers used. The mouse tail-tip fibroblast (TTF) showed a similar result (Fig. S2B). We preamplified single-cell DNA from human lung fibroblasts using telomere and Alu primers simultaneously for 20, 18, 16, 14, or 12 cycles and found the Ct value proportionally increased with decreasing cycle number from 18 to 14 (Fig. S1E). To determine the optimal pre-PCR cycle number, we prepared standard curves for telomere and reference genes by amplification of a series of dilutions of genomic DNA. The standard curves were quite linear within DNA concentrations from 1 ng/µL to 16 ng/µL for human cells (Fig. S2 C and D) and 0.375 ng/µL to 6 ng/µL for mouse cells (Fig. S2C). A comparison of the Ct value of standard DNA (Fig. S2D) and the Ct value after different numbers of pre-PCR cycles (Fig. S2E) indicated that pre-PCR cycle numbers from 16 to 18 were optimal; therefore, we chose the preamplification cycle number 17 for human cells and 16 for mouse cells, because the telomere length of mouse generally is longer than that of human and, expectedly, requires fewer cycles for amplification. We should point out that the T/R ratio obtained for mouse telomeres cannot be directly compared with T/R ratio for human telomeres. We suggest that a correlation of T/R ratio measured by SCT-pqPCR with TRF (kb) by Southern blot should be established initially using a cell population for each species and for each laboratory.

Correlation of Average Telomere Length Estimated by SCT-pqPCR and Telomere Length of Cell Populations by Conventional Methods.

Although the results above demonstrated that the telomere length of single cells can be measured by our assay, it was necessary to compare average telomere length calculated by averaging the results of single-cell measurements by SCT-pqPCR to established methods, including qPCR, Q-FISH, and TRF, which measure average telomere length of cell populations. Based on the above data, we compared telomere length of a population of cells measured by conventional qPCR (Fig. S1A) and Q-FISH (Fig. 3 and Fig. S3) to average telomere length of single cells measured by SCT-pqPCR in human cell lines F171, F200, HeLa S3, RuES2, U2OS, and SaOS2 (Table S1). As expected, the data showed that single-cell telomere length by SCT-pqPCR significantly correlated with average telomere length of cell populations measured by conventional qPCR and Q-FISH; the Pearson test produced P values of 0.001 and 0.006, respectively (Fig. 2 A and B). We also analyzed the correlation between SCT-pqPCR and TRF (Fig. S1B) in human cell lines (F171, F200, HeLa S3, RuES2, and SaOS2). The Pearson test again showed that the average of single-cell telomere lengths measured by SCT-pqPCR was highly correlated with absolute telomere length by TRF, with a P value of 0.015 (Fig. 2C). Interestingly, we found lower correlation between Q-FISH and TRF in these five cell lines (Fig. 2D). Presumably, the heterogeneity of single-cell telomere lengths in cancer cell lines exceeds that of normal cell lines. In addition, variations in telomere lengths between dividing and nondividing cells may bias Q-FISH results, because Q-FISH measures telomere lengths only of dividing cells capable of arrest at metaphase, but the TRF and qPCR methods measure average telomere length of all cells. This finding further supports the value of the SCT-pqPCR method for telomere measurement of any individual cells independent of their replication rate or potential.

Fig. 2.

Linear correlation of relative telomere length between single cells and their populations with various human cell lines. (A) Average telomere length in single cells as T/R ratio by SCT-pqPCR is significantly correlated with that of a cell population shown as the T/S ratio by regular qPCR. The Pearson test is applied. (B) Average telomere length in single cells by SCT-pqPCR is significantly correlated with quantitative telomere length by Q-FISH. (C) Average telomere length of single human cells by SCT-pqPCR is highly correlated with absolute telomere length of population cells by TRF. (D) Quantitative telomere length of metaphase cells by Q-FISH is correlated with the absolute 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 donor (F200), the pentagon represents human lung fibroblast from a 14-wk gestation (F171), the triangle represents SaOS2 cells, the square represents human ESC (RuES2), and the circle represent U2OS cell.

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.

To further validate our assay, we measured telomere lengths in dilutions of preamplified DNA of F171 and F200 after preamplifying DNA with target primers (telomere and reference gene Alu). When the input DNA was 2 ng, the relative telomere length in F171 was longer than that of F200 (Fig. S4), but when the DNA quantity decreased to 0.4 ng, there was no significant difference in telomere lengths between fibroblast F171 and F200, P > 0.05 (Fig. S4). Therefore, when the prepurified DNA drops below threshold value, one aliquot of the diluted DNA 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 6–7 pg DNA, 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, and F). 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 gene) (Fig. 3 B and E).

The limitation for the validation step above was that SCT-pqPCR and Q-FISH could not be performed for the same individual cells, and no somatic cells with identical telomere lengths could be reliably identified for this validation. To further validate the SCT-pqPCR assay, we measured telomere length in pairs of sister cells derived from two-cell mouse embryos and oocytes. Two-cell mouse embryos exhibit identical telomere lengths between sister blastomeres (22), and human polar bodies (PBs) show telomere lengths nearly identical to their matched MII oocytes by Q-FISH analysis (23). Telomere lengths were remarkably similar between sister blastomeres by SCT-pqPCR, P >> 0.1 (Fig. 4A and Table S2), although the one-way ANOVA (Tukey test) indicated that differences existed between pairs of sister cells from different embryos. Correlation analysis showed the telomere lengths between sister cells were proportional by the Pearson test (P = 0.005) (Fig. 4B). Telomere lengths between human oocytes (O) and their PBs demonstrated no significant differences by the paired samples t test, t = 0.603, P = 0.569 (Fig. 4C and Table S2). Occasional (e.g., PB1/O1, PB6/O6) PB and oocytes exhibited different telomere lengths, which could represent degeneration in telomere DNA or biological differences. Curiously telomere lengths of oocytes and PBs in patients 6 and 7 were remarkably longer than other patients by one-way ANOVA test, P < 0.001 (Fig. 4C). The telomere lengths of human oocytes correlated highly with the paired PB telomere lengths by liner correlation analysis (Fig. 4D). Measurements of telomere length in pairs of human oocytes and PBs by SCT-pqPCR agreed with our previous studies (23). Unlike sister cells of mouse two-cell embryos, telomere lengths between daughter cells of HeLa S3 frequently showed differences, although the correlation between telomere lengths in daughter cells was still high (Fig. 4 E and F, and Table S2).

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.

SCT-pqPCR Identifies Telomere Length and Its Heterogeneity in Single Cells from Various Cell Types.

We used SCT-pqPCR to measure telomeres of human lung fibroblast cell lines from different aged donors and passage numbers. When comparing telomere length in cell populations, the average telomere length of human fibroblast F171 passage number (P)16 (from 14-wk gestation) and F204 P14 (from a 35-y-old donor) was longer (P < 0.05) than that of human fibroblast F200 P7 (from a 71-y-old donor) by Q-FISH and conventional qPCR, but the average telomere length did not differ (P > 0.05) between F171 P16 and F204 P14 (Fig. S5). We then analyzed the single-cell telomere lengths between F171 P16 and F200 P7 by SCT-pqPCR. Remarkably, the telomere lengths of single cells differed in the same population of both F171 P16 and F200 P7 cells. Indeed, some single cells from F200 P7 had longer telomeres than F171 P16, as measured by SCT-pqPCR, a finding confirmed by Q-FISH analysis (Fig. 5 A and B and Fig. S3). The coefficient of variation (CV) showed single-cell telomere length in F200 P7 to be more heterogeneous than F171 P16 (Table 1). When human fibroblasts were continuously cultured (F171 P16 to P31 and F200 from P7 to P12), metaphase chromosome spreads were rarely available for analysis of cells at later passage, presumably because these cells had undergone senescence and failed to divide. Interestingly, SCT-pqPCR demonstrated increased variation in telomere length among cells at later passage compared with early passages (CV 0.486 in F171 P31 and 0.398 in F200 P12 vs. 0.169 in F171 P16 and 0.233 in F200 P7, respectively) (Fig. 5A and Table 1).

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 Q-FISH (B), shown as box-plot. The small circle represents an outlier; the asterisk represents extreme value. The result for each individual cell is shown in Fig. S3. F171 and F200 are human lung fibroblast from a 14-wk gestation and a 71-y-old donor, respectively. P16, P31, P7, and P12 refer to passage number. HeLa S3: human cancer cell. RuES2: human embryonic stem cell. U2OS and SaOS2: human osteosarcoma. Single-cell telomere length of human fibroblasts shortened with the age and passage number, and the heterogeneity of F171 and F200 increased with the passage. The later passage F171 P31 and F200 P12 were unavailable for metaphase telomere Q-FISH because of cell senescence and compromised division. (C) Telomere lengthening of single cells derived from mouse zygotes to four-cell embryos by SCT-pqPCR. Z, zygote; 2c, two-cell embryo; 4c, four-cell embryo. n = 6. (D) Analysis of average telomere length in different stage mouse embryos by Student Newman–Keuls test shows that telomere length elongates from zygote to four-cell embryo. n = 4. Bar is ± SD.

The above data show that telomere length varies among individual cells in a given population. We also analyzed telomere length of single cells in human ESC cultures (RuES2, telomerase activity-positive), cultured cancer cells (HeLa S3, telomerase activity-positive), and cultured osteosarcoma cells (U2OS and SaOS2, telomerase activity-negative) by SCT-pqPCR. As expected, average telomere length and variability among cells of a given population differed among these various cell types (Fig. 5A, and Fig. S3 A and C). The telomere lengths of HeLa S3 cells varied more than other cell types, with a CV of 0.294 (Table 1). Telomere lengths of single cells at metaphase in these cells (HeLa S3, Rues2, SaOS2, and U2OS) also showed extensive variations when measured by Q-FISH, confirming that single-cell telomere length varies within the same cell population (Fig. 5B, and Fig. S3 B and D). From the CV of different human cell lines as estimated by SCT-pqPCR and Q-FISH, the single-cell telomere lengths in cancer cell populations vary more than in early passage human fibroblast cell lines (Table 1). Similarly, fibroblasts from older people show more heterogeneity in telomere length than those of young people, and cells at later passages show telomere length variations more than those at earlier passages.

In addition, as a very delicate case, we also successfully measured the telomere lengths of individual cells from mouse zygote to four-cell embryos (Fig. 5C), and the average telomere length increased along with development of mouse embryos by SCT-pqPCR (Fig. 5D). Consistently, telomere length in developing mouse embryos was found to lengthen significantly from zygotes to the four-cell embryo stage (22).

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. See Fig. S6 for derivation of daughter cells and Table S3 for the sequence of primers used by qPCR.

Single-Cell Genomic DNA Amplification by PCR (pre-PCR) with Primers of Telomere and Reference Gene, and Product Purification.

Pre-PCR was performed using DNA Polymerase Hot Start Version (TAKARA). The reactions were set up by aliquoting 38 µL of master mix into the 0.2 mL PCR tubes each with 2 µL single-cell genomic DNA. Each reaction was set up with by 4 µL 10× PCR buffer, 4 µL 2.5 mM dNTP, 0.25 µL DNA polymerase, 1 µL each of telomere forward and reverse primer (10 µM), and 1 µL each of multicopy gene forward and reverse primer (10 µM) or single gene primer 36B4 (10 µM) and enough water to make up a 40-µL final volume. Thermal cycler reaction conditions were set at 94 °C for 5 min followed by different cycles of 94 °C for 15 s, 60 °C annealing for 30 s and extension at 72 °C for 30 s, with a final extension for 10 min at 72 °C. PCR products were purified following the protocol of the purification Kit (DNA clean and concentrator-5; Zymo Research). Finally the purified PCR products were eluted in 64 µL of double distilled water.

Q-PCR Assay for Average Telomere Measurement of a Cell Population.

Average telomere length was measured from total genomic DNA of human cell lines 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 addition, we chose multicopy genes (Alu and 18S rRNA) as reference genes and measured the average telomere length of different human cell lines. Each reaction included 10 µL 2× SYBR Green mix (Bio-Rad), 0.5 µL each of 10 µM forward and reverse primers, 4 µL molecular-filter water and 5 µL genomic DNA (7 ng/µL) to yield a 20-µL reaction. DNA samples were placed in adjacent three wells of a 96-well plate for telomere primers and reference gene primers, respectively. A Bio-Rad thermocycler (CFX system test) was used with reaction conditions of 95 °C for 10 min followed by 40 cycles of data collection at 95 °C for 15 s, 60 °C anneal for 30 s and 72 °C 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.

After thermal cycling was completed, the CFX manager software was used to generate standard curves and Ct values for telomere signals and reference gene signals. Here we used different reference genes for telomere length measurement, and each sample of DNA had one telomere signal and reference gene signal. The average telomere length was termed the T/R ratio instead of the T/S ratio.

Q-PCR Assay for Single-Cell Telomere Measurement.

Telomere length of single cells was measured by qPCR after pre-PCR, purification and aliquoting. The reaction was set up with SYBR Green I in 96-wells plates. The purified products of Pre-PCR for each single cell were aliquoted with 5 µL into each well of a 96-well plate. Three repeat reactions were performed for each sample plus each pair of primers. The final master mix of each well in the qPCR was 10 µL 2× SYBR Green mix, 0.5 µL each of forward and reverse primer (10 µM for Tel, Alu, B1, and 36B4) and 4 µL of molecular-filtered water. The qPCR conditions were the same as above. The results were analysis by CFX manager software and the relative telomere length of single cells was calculated by the T/R ratio.

Telomere Q-FISH.

Telomere Q-FISH and quantification were performed as described previously (11, 22).

Southern Blot Analysis of TRF.

Telomere restriction fragment analysis was performed as described by using the TeloTAGGG Telomere Length Assay Kit (Roche) according to the protocol provided by the manufacturer. The mean telomere length was calculated using the following (26): TRF = ∑(ODi)/∑(ODi/Li), where ODi is the chemiluminescent signal and ODi/Li is the length of the TRF at position.

Data Statistics.

All of the data statistics were obtained by SPSS 13.0 software. The P value for comparison of two groups was derived from the independent-samples t test. The multiple groups’ statistical data were analyzed with one-way ANOVA. The frequency of telomere distribution was compared by a nonparametric test. The correlation analysis was performed by the Pearson correlation test; α is set at 0.05 for all tests.

Supplementary Material