Here, we describe in detail the protocol for quantifying telomere length using non-radioactive chemiluminescent detection, with a focus on the optimization of various performance parameters of the TAGGG telomere length assay kit, such as buffer quantities and probe concentrations.
Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening occurs due to a problem with end replication and the absence of the telomerase enzyme, which is responsible for maintaining telomere length. Interestingly, telomeres also shorten in response to various internal physiological processes, like oxidative stress and inflammation, which may be impacted due to extracellular agents like pollutants, infectious agents, nutrients, or radiation. Thus, telomere length serves as an excellent biomarker of aging and various physiological health parameters. The TAGGG telomere length assay kit is used to quantify average telomere lengths using the telomere restriction fragment (TRF) assay and is highly reproducible. However, it is an expensive method, and because of this, it is not employed routinely for large sample numbers. Here, we describe a detailed protocol for an optimized and cost-effective measurement of telomere length using Southern blots or TRF analysis and non-radioactive chemiluminescence-based detection.
Telomeres are the repetitive DNA sequences present at the end of chromosomes. They have tandem repeats of TTAGGG and maintain genome integrity by protecting the chromosome from both fraying and the end replication problem, which means that part of the 3' overhang is unable to be replicated by DNA polymerase1,2. Short telomeres lead to chromosomal abnormalities in cells, due to which cells become permanently arrested in a stage called replicative senescence3. Short telomeres also cause a host of other problems, such as mitochondria dysfunction4,5 and cell dysfunction.
DNA telomeric repeats are lost as and when the cell divides, with an average loss of 25 to 200 bp per year6, resulting in cellular senescence after a certain number of divisions6. Aging is associated with a higher frequency of comorbidities, which is marked by a shortening in telomere length7. Telomere restriction fragment (TRF) analysis, as described by Mender, is a very expensive method8. Because of this, it is not implemented while quantifying telomere length in most studies.
Presently, the majority of epidemiological studies employ quantitative polymerase chain reaction (qPCR)-based measurements of telomere length. However, the qPCR-based method is a relative measurement method, as it measures the ratio between telomeres and single-copy gene amplification products, and not absolute telomere length. Telomere length measurement using the TRF protocol is the gold standard method, as it can measure telomere length distribution in the sample and measurements can be expressed in absolute values in kilobases (kb). However, its use is limited because it is cumbersome, labor-intensive, and costly. Here, we present an optimized protocol for telomere length measurement using chemiluminescence-based TRFs.
TRF analysis includes seven major steps: 1) culturing of cells for genomic DNA extraction, 2) genomic DNA extraction using the phenol:chloroform:isoamylalcohol (P:C:I) method, 3) restriction digestion of genomic DNA, 4) agarose gel electrophoresis, 5) Southern blotting of the restriction digestion DNA fragment, 6) hybridization and detection via chemiluminescence-the immobilized telomere probe is visualized by a highly sensitive chemiluminescent substrate for alkaline phosphatase, disodium 2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,2′-(5-chlorotricyclo[3.3.1.13.7]decan])-4-yl]-1-phenyl phosphate (CDP-Star)-and 7) analysis for obtaining mean telomere length and range information from these telomeric smears.
NOTE: See the Table of Materials for details about all reagents used in the protocol below. Table 1 enlists lab-made reagents along with optimized volumes and Table 2 shows working concentrations of commercially available reagents.
1. Cell culture
2. Genomic DNA isolation
3. Digestion of genomic DNA
4. Agarose gel electrophoresis
5. Southern blotting
6. Hybridization and chemiluminescence detection
7. Analysis
The extracted genomic DNA (gDNA), which was run on a 1% agarose gel, showed good integrity, as shown in Figure 1B, indicating that the sample could be used for further downstream processing of TRFs. The TRF assay was then carried out by the modifying the volumes of solutions required at each step (see Table 1 and Table 2). The TRF signal was clearly visible (Figure 3). Thus, by modifying the solution volumes and concentrations, more samples could be processed without any negative effect on the results, and the telomere length could be determined successfully using freely available software such as Telotool10.
Figure 1: Isolation and quality check of genomic DNA. (A) Three distinct separation phases obtained upon centrifugation after adding phenol:chloroform:isoamyl alcohol. The upper aqueous layer contains the gDNA, the interphase contains the proteins, and the lower, organic phase contains the degraded RNA, cell debris, and lipids. (B) An image of a 1% agarose gel showing intact undigested gDNA. Lane 1 shows a 1 kb ladder and lane 2 shows undigested gDNA of the cancer cell line A2780. Abbreviation: gDNA = genomic DNA. Please click here to view a larger version of this figure.
Figure 2: Illustration of Southern blotting transfer setup. Representative diagram showing the system assembly for the transfer of telomere repeat smears from the gel to the nylon membrane by capillary action. Please click here to view a larger version of this figure.
Figure 3: Chemiluminescence detection of TRFs post-Southern blotting and hybridization. Blot showing a range of telomeric repeats as a smear in lane 2. Lane 1 shows a molecular marker, with the molecular weights (kb) of bands indicated on left side. Please click here to view a larger version of this figure.
Table 1: List of reagents used in this protocol. The table contains reagents prepared in the lab along with their storage details, the recommended usage of the TAGGG telomere length assay kit reagents, and their modified usage volumes, as per the optimization in this protocol. Please click here to download this Table.
Table 2: List of commercially available reagents with modified usage instructions. The table contains commercially available reagents along with different dilutions, their storage details, and their recommended usage by the TAGGG telomere length assay kit and modified usage volumes, as per the optimization in this protocol. Please click here to download this Table.
We describe a detailed procedure for a non-radioactive, chemiluminescence-based method for telomere length measurement using Southern blotting. The protocol has been tested to allow the judicious use of several reagents with no compromise on the quality of results. The prehybridization and hybridization buffer can be reused up to five times. Enzyme concentration can vary between 10-20 U per 1.5-2 µg of genomic DNA without affecting the results. Several other kit components, such as the DIG-labeled molecular weight marker and hybridization probe, can be used at lower concentrations than recommended. The optimized volumes are indicated in Table 2. We have tested them at half the recommended volumes/concentrations and found optimal performance. Following these steps reduces the cost per sample tremendously, thus enabling researchers to measure telomere length using this method at a larger scale.
There are several published TRF protocols. We have optimized the commercially available kit protocol to make it cost-effective. This protocol is different from some of the published protocols as it does not use radioactivity11,12. It is also relatively simple, as it uses the kit components rather than preparing in-house reagents, particularly the telomere probe13.
If the final image is patchy, then the membrane has partially dried during incubation. To resolve this, one should either increase the solution volume or agitate at a higher speed. If there are no smears visible on the blot, then there might have been a problem during the Southern transfer. In addition, there could have been a problem in the DNA quantity or quality.
There are some limitations to this method. First, genomic DNA extraction and quantification takes upward of 4 days based on the source of the DNA14. Higher molecular weight DNA takes a longer time to rehydrate and homogenize, making quantification and accurate pipetting difficult. Second, the TRF protocol is long (2 days minimum) and requires skilled lab workers to implement. It is also an expensive method due to the reagents and the quantities required. The TRF protocol also measures the average telomere length in each sample, as opposed to the shortest telomere length that TESLA measures or the telomere length per cell that flow cytometry-based methods provide15. Another limitation of TRF analysis using the enzymes used here is an overestimation of telomere length, as these enzymes do not have a restriction site in the sub-telomeric regions15.
However, despite the limitations, there are reasons why this method is still considered the gold standard of telomere length measurement. Telomere lengths are rendered accurately in absolute values-in kilobase pairs (kbp).
This method can be used to measure the average telomere length in a variety of cell types, from cell lines16,17 to buffy coat pellets18,19. This makes it a robust method to use in several types of research studies. It can also be used in large-scale epidemiological studies as well as in studies where cell-based therapeutics are being developed.
The authors have nothing to disclose.
We would like to acknowledge Ms. Prachi Shah for helping us initially with the protocol optimization. We would like to thank Dr. Manoj Garg for providing the A2780 ovarian cancer cell line. EK is supported by a Research Grant from the Department of Biotechnology (No. BT/RLF/Re-entry/06/2015), Department of Science and Technology (ECR/2018/002117), and NMIMS Seed Grant (IO 401405).
Cell Line | |||
A2780 (Ovarian adenocarcinoma cell line) | Received as a gift | ||
Equipment | |||
ChemiDoc XRS+ (for imaging and UV cross linking) | Biorad | Universal hood II (721BR14277) | |
Nanodrop (Epoch 2) | Biotek | EPOCH2 | |
Software | |||
TeloTool | Version 1.3 | ||
Materials | |||
Acetic Acid | Molychem | 64-19-7 | |
Agarose | MP | 180720 | |
Amphotericin B | Gibco, ThermoFisher Scientific, USA | 15240062 | |
DMEM | HyClone, Cytiva, USA | SH30243.01 | |
Ethylenediamine tetraacetic acid | Molychem | 6381-92-6 | |
HI FBS | Gibco, ThermoFisher Scientific, USA | 10270106 | |
HCl | Molychem | 76-47-01-0 | |
NaCl | Molychem | 7647-14-5 | |
NaOH | Molychem | 1310-73-2 | |
Nylon membrane | Sigma | 11209299001 | |
Penicillin | Gibco, ThermoFisher Scientific, USA | 15240062 | |
Sodium dodecyl sulfate | Affymetrix | 151-21-3 | |
Streptomycin | Gibco, ThermoFisher Scientific, USA | 15240062 | |
Tris | BIORAD | 77-86-1 | |
Tris HCl | Sigma Aldrich | 1185-53-1 | |
Whatman paper | GE healthcare lifesciences | 1001-917 | |
Reagents | |||
1 kb ladder | NEB | N3232S | |
20x SSC | Invitrogen | 15557-036 | |
Anti DIG AP | Telo TAGGG Telomere Length Assay kit | 12209136001 | |
Blocking solution 10x | Telo TAGGG Telomere Length Assay kit | 12209136001 | |
Cutsmart Buffer | NEB | B6004 | |
Detection buffer 10x | Telo TAGGG Telomere Length Assay kit | 12209136001 | |
Dig easy hyb | Telo TAGGG Telomere Length Assay kit | 12209136001 | |
Digestion Buffer | Telo TAGGG Telomere Length Assay kit | 12209136001 | |
Hinf 1 | Telo TAGGG Telomere Length Assay kit | 12209136001 | |
Hinf 1 (alternative to kit) | NEB | R0155T | |
Loading Dye | BIOLABS | N3231S | |
Maleic acid buffer 10x | Telo TAGGG Telomere Length Assay kit | 12209136001 | |
Molecular marker | Telo TAGGG Telomere Length Assay kit | 12209136001 | |
Probe | Telo TAGGG Telomere Length Assay kit | 12209136001 | |
Rsa 1 | Telo TAGGG Telomere Length Assay kit | 12209136001 | |
Rsa 1 (alternative to kit) | NEB | R0167L | |
Substrate | Telo TAGGG Telomere Length Assay kit | 12209136001 | |
Wash buffer | Telo TAGGG Telomere Length Assay kit | 12209136001 |