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Abstract

Introduction

Protocol

Representative Results

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Acknowledgements

Materials

References

Cancer Research

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Published: April 21st, 2023

DOI:

10.3791/65288

1Sunandan Divatia School of Science, SVKM's NMIMS Deemed-to-be-University
* These authors contributed equally

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,

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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

  1. Maintain cells whose telomere length is to be measured (used here were A2780 cells, which is an ovarian adenocarcinoma cell line) in Dulbecco's modified eagle medium .......

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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, .......

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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 m.......

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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).

....

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Name Company Catalog Number Comments
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

  1. Greider, C. W. Telomere length regulation. Annual Review of Biochemistry. 65, 337-365 (1996).
  2. Valdes, A. M., et al. Obesity, cigarette smoking, and telomere length in women. Lancet. 366 (9486), 662-664 (2005).
  3. Allsopp, R. C., et al. Telomere length predicts replicative capacity of human fibroblasts. Proceedings of the National Academy of Sciences. 89 (21), 10114-10118 (1992).
  4. Epel, E. S., et al. Accelerated telomere shortening in response to life stress. Proceedings of the National Academy of Sciences. 101 (49), 17312-17315 (2004).
  5. Canela, A., Vera, E., Klatt, P., Blasco, M. A. High-throughput telomere length quantification by FISH and its application to human population studies. Proceedings of the National Academy of Sciences. 104 (13), 5300-5305 (2007).
  6. Révész, D., Milaneschi, Y., Verhoeven, J. E., Penninx, B. W. Telomere length as a marker of cellular aging is associated with prevalence and progression of metabolic syndrome. The Journal of Clinical Endocrinology and Metabolism. 99 (12), 4607-4615 (2014).
  7. Rizvi, S., Raza, S. T., Mahdi, F. Telomere length variations in aging and age-related diseases. Current Aging Science. 7 (3), 161-167 (2014).
  8. Mender, I., Shay, J. W. Telomere restriction fragment (TRF) analysis. Bio-Protocol. 5 (22), e1658 (2015).
  9. Zhu, Y., Liu, X., Ding, X., Wang, F., Geng, X. Telomere and its role in the aging pathways: telomere shortening, cell senescence and mitochondria dysfunction. Biogerontology. 20 (1), 1-16 (2019).
  10. Göhring, J., Fulcher, N., Jacak, J., Riha, K. TeloTool: a new tool for telomere length measurement from terminal restriction fragment analysis with improved probe intensity correction. Nucleic Acids Research. 42 (3), 21 (2014).
  11. Jenkins, F. J., Kerr, C. M., Fouquerel, E., Bovbjerg, D. H., Opresko, P. L. Modified terminal restriction fragment analysis for quantifying telomere length using in-gel hybridization. Journal of Visualized Experiments. (125), e56001 (2017).
  12. Fojtová, M., Fajkus, P., Sováková, P. P., Fajkus, J. Terminal restriction fragments (TRF) method to analyze telomere lengths. Bio-protocol. 5 (23), e1671 (2015).
  13. Kimura, M., et al. Measurement of telomere length by the Southern blot analysis of terminal restriction fragment lengths. Nature Protocols. 5 (9), 1596-1607 (2010).
  14. Trigodet, F., et al. High molecular weight DNA extraction strategies for long-read sequencing of complex metagenomes. Molecular Ecology Resources. 22 (5), 1786-1802 (2022).
  15. Lai, T. P., Wright, W. E., Shay, J. W. Comparison of telomere length measurement methods. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 373 (1741), 20160451 (2018).
  16. Mochida, A., et al. Telomere size and telomerase activity in Epstein-Barr virus (EBV)-positive and EBV-negative Burkitt's lymphoma cell lines. Archives of Virology. 150 (10), 2139-2150 (2005).
  17. Gupta, N., et al. Replicative senescence, telomere shortening and cell proliferation rate in Gaddi goat's skin fibroblast cell line. Cell Biology International. 31 (10), 1257-1264 (2007).
  18. Michaeli, J., et al. Leukocyte telomere length correlates with extended female fertility. Cells. 11 (3), 513 (2022).
  19. Lesmana, A., et al. Continuous reference intervals for leukocyte telomere length in children: the method matters. Clinical Chemistry and Laboratory Medicine. 59 (7), 1279-1288 (2021).

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