In this article, a detailed protocol for quantifying telomere length using a modified terminal restriction fragment analysis is discussed that provides fast and efficient direct measurement of telomere length. This technique can be applied to a variety of cell sources of DNA for quantifying telomere length.
There are several different techniques for measuring telomere length, each with their own advantages and disadvantages. The traditional approach, Telomere Restriction Fragment (TRF) analysis, utilizes a DNA hybridization technique whereby genomic DNA samples are digested with restriction enzymes, leaving behind telomere DNA repeats and some sub-telomeric DNA. These are separated by agarose gel electrophoresis, transferred to a filter membrane and hybridized to oligonucleotide probes tagged with either chemiluminescence or radioactivity to visualize telomere restriction fragments. This approach, while requiring a larger quantity of DNA than other techniques such as PCR, can measure the telomere length distribution of a population of cells and allows measurement expressed in absolute kilobases. This manuscript demonstrates a modified DNA hybridization procedure for determining telomere length. Genomic DNA is first digested with restriction enzymes (that do not cut telomeres) and separated by agarose gel electrophoresis. The gel is then dried and the DNA is denatured and hybridized in situ to a radiolabeled oligonucleotide probe. This in situ hybridization avoids loss of telomere DNA and improves signal intensity. Following hybridization, the gels are imaged utilizing phosphor screens and the telomere length is quantified using a graphing program. This procedure was developed by the laboratories of Drs. Woodring Wright and Jerry Shay at the University of Texas Southwestern1,2. Here, we present a detailed description of this procedure, with some modifications.
Telomeres, situated at the ends of chromosomes, are nucleotide repeats of the sequence TTAGGG (on human chromosomes) that play a critical role in protecting cellular genetic information3,4,5. Telomeres are several thousand base pairs in length and serve to protect the integrity of the rest of the chromosome during DNA replication. Replication of chromosomes is not perfect resulting in the ends of the chromosome not being completely copied. This inefficiency in replication is termed the "end-replication problem" and results in loss of some of the telomere repeats during each round of replication6,7. Telomere length can be restored after cell division by telomerase, an enzyme that replaces the lost telomere sequences8,9. Telomerase, however, is not activated in most human somatic cells and as a result, over time, telomeres will shorten, eventually resulting in cellular senescence10. Due to telomere shortening, telomeres are regarded as a biomarker of aging and risk of age-related diseases11,12. Additionally, telomere length can be affected by genetic, environmental, and lifestyle factors and other stimuli such as oxidative stress, indicating that telomere length can play a role as a biomarker in toxicological, epidemiological and behavioral studies13,14,15.
This article demonstrates a technique for measuring telomere length using a modified terminal restriction fragment (TRF) analysis adapted from Mender and Shay2 and Kimura et al.16, and similar to Herbert, Shay and Wright1 and Haussmann and Mauck17. Traditionally, TRF analysis involves a Southern blot procedure. Southern blots are considered the "gold-standard" for studying telomere length and are actively used for examining leukocyte telomere length in epidemiology studies18. This TRF analysis uses digested genomic DNA leaving behind the telomere repeats. The DNA fragments are then separated by gel electrophoresis, denatured and transferred to a nitrocellulose membrane. The telomere sequences are hybridized to a telomere oligonucleotide probe. Rather than transferring the separated telomeres to a membrane, other TRF techniques use in-gel hybridization techniques with and without denaturation of the DNA. The inclusion of denaturation is important when considering the detection of interstitial telomeres, which have been reported in some species19. Procedures that lack denaturing steps only detect the single stranded DNA overhangs at terminal telomeres and will not bind to interstitial telomere sequences18.
Besides TRF analysis, there are several other techniques for measuring telomere length that each have their own advantages and disadvantages. The Flow-FISH technique can measure mean telomere length of individual cells of a distinct cell type using flow cytometry16,20. While it can accurately tag telomeres with highly specific probes and reduce human error through automation, Flow-FISH is expensive, less efficient and requires fresh samples and a highly-skilled technician, limiting its ability for epidemiology studies16. In addition, this method does not account for potential changes in the number of chromosomes in the cell population. Another technique, qPCR, is widely accepted as a means of measuring telomere length for epidemiology studies16 due to its high-throughput, low-cost and requirement for small amounts of DNA compared to other methods. However, qPCR can only measure average telomeric DNA content relative to a single copy gene and is, thus, an indirect estimate of average telomere length. It also yields a high coefficient of variance in comparison studies, compared to southern blots, leading to questionable accuracy21.
The TRF procedure has its own deficiencies that limit its use for some studies. TRF techniques require a large amount of DNA compared to other methods (between 2 and 3 µg per sample). This limits the technique's applications to examining telomere length of clinical samples for which sufficient genomic DNA can be collected, and cannot be used on degraded DNA samples. Additionally, the TRF analysis is costly and labor intensive, requiring 4-5 days to yield results. However, the TRF yields a low coefficient of variance granting its reproducibility. While this protocol is different than the traditional Southern blot (utilizing in situ gel hybridization), the two techniques are fundamentally the same.
The technique presented here is a combination of a Southern blot and in-gel hybridization; associating the DNA denaturing step from Southern blots with the in-gel hybridization. This combination has the added benefit of improved probe signal strength compared to the Southern blot and has consistently yielded quantifiable results within our lab. Additionally, the use of radioactive probes rather than chemiluminescent probes yields higher signal intensity and allows for visualization and quantification with a phosphorimager, making the analyses of the TRF user-friendly.
1. Genomic DNA Extraction
2. DNA Integrity Assessment
NOTE: This portion of the protocol outlines a DNA integrity assessment using a gel electrophoresis system with a 11 cm x 14 cm gel. Other systems and gel sizes can also be used, but the voltage and DNA migration time may vary.
3. Genomic DNA Digestion
4. Genomic DNA Gel Electrophoresis
NOTE: This portion of the protocol outlines a procedure for using a 20 cm x 25 cm gel electrophoresis system. Different gel electrophoresis systems can be used, but several variables may need to be modified including voltage, time of migration and amount of electrophoresis buffer added to the system in order to yield optimal results.
5. Gel Fluorescent Imaging and Hybridization
6. Hybridization
7. Gel Washing, Phosphor Screen Exposure, and Radio Imaging
8. Telomere Length Quantification
Three concentrations of DNA (1, 2 and 3 µg) isolated from the cell line BJ-hTERT (telomerase immortalized human foreskin fibroblasts)22 and human peripheral blood mononuclear cells (PBMCs) were analyzed for telomere length. Table 1 shows the lane assignments for each DNA sample. Figure 1 shows a nucleic acid fluorescent stain of the gel. The molecular weight standards are clearly visible. The distance from the top of the gel to each molecular weight standard was determined (Figure 2). Figure 3 shows the phosphorimage of the gel following hybridization with the telomere probe. The telomere bands are shown as smears in each lane. Grids of 150 boxes were calculated for each lane plus one background lane (Figure 4). The areas of analysis corresponding to the areas of the gel containing the telomere bands for each set of samples is shown in Figure 5. Separate background lanes were selected for each set of samples to ensure that the background intensities were similar for each sample set (lanes marked B in Figure 5). Table 2 shows the telomere restriction fragment lengths for each sample. The average telomere length in the hTERT immortalized cell line (BJ-hTERT) is higher than that seen in human PBMCs (compare lanes 2, 4 and 6 with lanes 10, 12, and 14 in Figure 3). These results also demonstrate that the amount of genomic DNA analyzed in each lane is critical to obtaining a detectable signal following hybridization. For samples with telomeres that are fairly uniform (i.e., the BJ-hTERT cell line) as little as 1 µg was detectable using this procedure (although the signal was very faint). However, for samples with telomeres that have a larger variance (i.e., human PBMCs), the minimum amount of DNA providing a reliable signal is 2 µg.
Figure 1: Green Fluorescent Nucleic Acid Stained Gel. Phosphorimage of the dried agarose gel stained with green fluorescent nucleic acid stain showing the location of the DNA ladder bands (lanes 1 and 8). Please click here to view a larger version of this figure.
Figure 2: Determination of the Marker Band Migration Distance. Blue arrows indicate the distance measured from the top of the gel to each DNA ladder band. Please click here to view a larger version of this figure.
Figure 3: Phosphorimage of DNA Samples Probed with the Telomere Probe. Lanes 2, 4 and 6 contain BJ-hTERT DNA (1, 2, and 3 µg, respectively). Lanes 10, 12 and 14 contain human PBMC DNA (1, 2 and 3 µg, respectively). Please click here to view a larger version of this figure.
Figure 4: Grid Location. Screenshot showing the location of 150 count grids for each analyzed lane. Please click here to view a larger version of this figure.
Figure 5: Areas of Analysis. Phosphorimage of telomere bands showing the selected areas of analysis for each set of samples. Lanes 2, 4 and 6 contain BJ-hTERT DNA; lanes 10, 12 and 14 contain human PBMC DNA. B = background lanes. Please click here to view a larger version of this figure.
Lane | Sample |
1 | DNA Ladder |
2 | BJ-hTERT 1µg |
3 | Blank |
4 | BJ-hTERT 2 µg |
5 | Blank |
6 | BJ-hTERT 3µg |
7 | Blank |
8 | DNA Ladder |
9 | Blank |
10 | PBMCs 1µg |
11 | Blank |
12 | PBMCs 2µg |
13 | Blank |
14 | PBMCs 3µg |
DNA Samples separated on 0.6% agarose gel |
Table 1: DNA Agarose Sample Assignments.
Sample (Lane) | ∑(Inti) | ∑(Inti/MWi) | Average Telomere Length = ∑(Inti)/∑(Inti/MWi) |
BJTert 1ug (1) | 268691 | 20596 | 13.04578559 |
BJTert 2ug (2) | 1357000 | 103204 | 13.14871517 |
BJTert 3ug (3) | 1962000 | 148411 | 13.22004434 |
PBMC 1ug (4) | -416337 | -142585 | 2.91992145 |
PBMC 2ug (5) | 286653 | 62845 | 4.561269791 |
PBMC 3ug (6) | 1880000 | 524123 | 3.586944286 |
Table 2: Average Telomere Length Calculations.
Following electrophoresis, the genomic DNA smear is higher than 800 bp. This can occur if the restriction enzymes are faulty or if additional enzymes (as described above) are needed. A second possible explanation is that the protein is still attached to the DNA. When determining the DNA concentration by spectrophotometer, the 260/280 ratio should be around 1.8. If this ratio is <1.5, there is protein contamination which can interfere with restriction enzyme digestion. Either the DNA sample would need to be re-isolated, or the protein would need to be removed with a proteinase K digestion and phenol:chloroform extraction followed by ethanol precipitation. The use of a commercial DNA isolation kit routinely results in DNA with a 260/280 ratio of 1.8-1.85.
Following the exposure of the gel to a phosphor screen, there are no telomere fragments detected. This can result from a poor radio-labelled telomere probe, improper hybridization conditions or insufficient quantity of starting genomic DNA. When preparing the telomere probe, at the end of the centrifugation of the G-25 column, there should be readily detectable radioactivity in the flow-through (representing the radio-labelled telomere probe). If radioactivity is not detected, then there was a problem with the preparation of the probe. It is also important to ensure that the hybridization temperature does not exceed 42 °C (to prevent melting of the probe:template complex) and that the hybridization buffer completely immerses the gel in the hybridization chamber. The minimum amount of genomic DNA that provides detectable telomeres is 2.0-3.0 µg. Smaller amounts of starting material can result in very weak to no signal following hybridization.
This protocol requires 2-3 µg for each telomere length determination. If the samples are to be run in duplicate or triplicate, then 6-12 µg of DNA is required. This prevents this procedure from being used on samples with limited DNA quantity. The procedure is fairly laborious requiring 3-4 days to complete. The number of samples analyzed at one time is limited to the number of gel electrophoresis rigs and the size of the agarose gel comb. As a result, it is less than ideal for studies employing large numbers of samples (such as epidemiological studies).
DNA hybridization techniques of telomeres separated by gel electrophoresis remains the gold standard for determining telomere length. No other procedure directly measures telomere length resulting in actual telomere sizes.
There are several critical steps in this procedure. First, the DNA sample must have good integrity, i.e., not sheared. DNA that is sheared can result in false telomere length results. It is also important that the DNA digestions be complete. The digestion of genomic DNA should result in a DNA smear of 800 bp or less2. To ensure proper digestion, some procedures have described a total of 6 restriction enzymes (rather than just RsaI and Hinf1 as described in this procedure). The additional restriction enzymes include HhaI, MspI, HaeIII and AluI2. A second critical step is allowing the agarose gel to run long enough to get proper separation of the digested DNA. If there is better separation, then there is better precision in the analyses. The gel run time should be long enough so that DNA fragments representing 1 Kbp are located at the bottom of the gel (Figure 1). Depending on the gel size and the voltage of the electrophoresis, this can require 12 to 24 h. A third critical step is the hybridization. There must be sufficient hybridization buffer in the chamber tube to immerse the entire gel when the tube is horizontal. Insufficient levels of hybridization buffer can result in uneven hybridization of the telomere probe.
The authors have nothing to disclose.
The authors acknowledge the support of the University of Pittsburgh Cancer Institute Biobehavioral Oncology shared facility that is supported in part by award P30CA047904.
Biologics | |||
Rsa1, restriction enzyme | New England Biolabs, Inc | R0167S | 10,000 U/mL, DNA digestion. Comes with Cutsmart Buffer (digestion buffer) |
Hinf1, restriction enzyme | New England Biolabs, Inc | R0155S | 10,000 U/mL, DNA digestion. Comes with Cutsmart Buffer (digestion buffer) |
Seakem GTG Agarose | Lonza | 50071 | electrophoresis grade agarose |
Optikinase | affymetrix | 78334X 500 UN | T4 Polynucleotide Kinase |
SYBR Green Nucleic Acid Stain I | ThermoFisher Scientific | S7567 | Syber Green |
exactGene DNA Ladder 24- kB | Fisher Scientific | BP2580100 | |
C-Strand Probe (3'-(G3AT2)4-'5) | Integrated DNA Technologies | Custom ordered DNA oligonucleotide | |
Gamma-ATP-P32 | Perkin Elmer | BLU502Z | Radioisotope |
Qiagen Blood and Cell Culture DNA Mini Kit | Qiagen | 13323 | DNA extraction kit |
GE Illustra Microspin G-25 column | GE Healthcare Life Sciences | 27-5325-01 | For purification of readioactive oligo probe |
Ficoll 400 | non-ionic synthetic polymer | ||
Equipment/Software | |||
Owl A5 Gel electrophoresis system (20 cm x 25 cm) | ThermoFisher Scientific | A5 | Gel electrophoresis |
Horizon 11.4 Gel electrophoresis system (11 cm x 14 cm) | Corel Life Sciences | 11068020 | Gel electrophoresis |
Nylon mesh, 50, 12" x 12" | Ted Pellam, Inc | 41-12105 | |
GE Storage Phosphor Screens | GE Healthcare Life Sciences | 28-9564-75 | |
Phosphor Screen Exposure Cassette | GE Healthcare Life Sciences | 63-0035-44 | |
Isotemp Hybridzation Incubator | Fisher Scientific | 13-247-20Q | |
Cylindrical hybridization tubes | Fisher Scientific | 13-247-300 | |
The Belly Dancer orbital shaker | Sigma-aldrich | Z768499-1EA | Orbital shaker |
Typhoon 9400 | ABI | For imaging of gels and phosphor screens | |
GraphPad Prism 6 | GraphPad | Version 6.05 | Graphing Program |
Microsoft Excel | Microsoft | Office 365 | Spreadsheet program |
Nanodrop | Thermo Scientific | Nanodrp 2000 | Spectrophotometer |