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).

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
<ol>
	<li>Maintain cells whose telomere length is to be measured (used here were A2780 cells, which is an ovarian adenocarcinoma cell line) in Dulbecco&#39;s modified eagle medium ...

Quantifying Telomere Length Using Non-Radioactive Chemiluminescence Detection

<ol>
	<li>Greider, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+length+regulation.">Telomere length regulation.</a> Annual Review of Biochemistry. 65, 337-365 (1996).</li><li>Valdes, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity,+cigarette+smoking,+and+telomere+length+in+women.">Obesity, cigarette smoking, and telomere length in women.</a> Lancet. 366 (9486), 662-664 (2005).</li><li>Allsopp, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+length+predicts+replicative+capacity+of+human+fibroblasts.">Telomere length predicts replicative capacity of human fibroblasts.</a> Proceedings of the National Academy of Sciences. 89 (21), 10114-10118 (1992).</li><li>Epel, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+telomere+shortening+in+response+to+life+stress.">Accelerated telomere shortening in response to life stress.</a> Proceedings of the National Academy of Sciences. 101 (49), 17312-17315 (2004).</li><li>Canela, A., Vera, E., Klatt, P., Blasco, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+telomere+length+quantification+by+FISH+and+its+application+to+human+population+studies.">High-throughput telomere length quantification by FISH and its application to human population studies.</a> Proceedings of the National Academy of Sciences. 104 (13), 5300-5305 (2007).</li><li>R&#233;v&#233;sz, D., Milaneschi, Y., Verhoeven, J. E., Penninx, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+length+as+a+marker+of+cellular+aging+is+associated+with+prevalence+and+progression+of+metabolic+syndrome.">Telomere length as a marker of cellular aging is associated with prevalence and progression of metabolic syndrome.</a> The Journal of Clinical Endocrinology and Metabolism. 99 (12), 4607-4615 (2014).</li><li>Rizvi, S., Raza, S. T., Mahdi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+length+variations+in+aging+and+age-related+diseases.">Telomere length variations in aging and age-related diseases.</a> Current Aging Science. 7 (3), 161-167 (2014).</li><li>Mender, I., Shay, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+restriction+fragment+(TRF)+analysis.">Telomere restriction fragment (TRF) analysis.</a> Bio-Protocol. 5 (22), e1658 (2015).</li><li>Zhu, Y., Liu, X., Ding, X., Wang, F., Geng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+and+its+role+in+the+aging+pathways:+telomere+shortening,+cell+senescence+and+mitochondria+dysfunction.">Telomere and its role in the aging pathways: telomere shortening, cell senescence and mitochondria dysfunction.</a> Biogerontology. 20 (1), 1-16 (2019).</li><li>G&#246;hring, J., Fulcher, N., Jacak, J., Riha, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TeloTool:+a+new+tool+for+telomere+length+measurement+from+terminal+restriction+fragment+analysis+with+improved+probe+intensity+correction.">TeloTool: a new tool for telomere length measurement from terminal restriction fragment analysis with improved probe intensity correction.</a> Nucleic Acids Research. 42 (3), 21 (2014).</li><li>Jenkins, F. J., Kerr, C. M., Fouquerel, E., Bovbjerg, D. H., Opresko, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+terminal+restriction+fragment+analysis+for+quantifying+telomere+length+using+in-gel+hybridization.">Modified terminal restriction fragment analysis for quantifying telomere length using in-gel hybridization.</a> Journal of Visualized Experiments. (125), e56001 (2017).</li><li>Fojtov&#225;, M., Fajkus, P., Sov&#225;kov&#225;, P. P., Fajkus, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terminal+restriction+fragments+(TRF)+method+to+analyze+telomere+lengths.">Terminal restriction fragments (TRF) method to analyze telomere lengths.</a> Bio-protocol. 5 (23), e1671 (2015).</li><li>Kimura, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+telomere+length+by+the+Southern+blot+analysis+of+terminal+restriction+fragment+lengths.">Measurement of telomere length by the Southern blot analysis of terminal restriction fragment lengths.</a> Nature Protocols. 5 (9), 1596-1607 (2010).</li><li>Trigodet, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+molecular+weight+DNA+extraction+strategies+for+long-read+sequencing+of+complex+metagenomes.">High molecular weight DNA extraction strategies for long-read sequencing of complex metagenomes.</a> Molecular Ecology Resources. 22 (5), 1786-1802 (2022).</li><li>Lai, T. P., Wright, W. E., Shay, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+telomere+length+measurement+methods.">Comparison of telomere length measurement methods.</a> Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 373 (1741), 20160451 (2018).</li><li>Mochida, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+size+and+telomerase+activity+in+Epstein-Barr+virus+(EBV)-positive+and+EBV-negative+Burkitt's+lymphoma+cell+lines.">Telomere size and telomerase activity in Epstein-Barr virus (EBV)-positive and EBV-negative Burkitt's lymphoma cell lines.</a> Archives of Virology. 150 (10), 2139-2150 (2005).</li><li>Gupta, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replicative+senescence,+telomere+shortening+and+cell+proliferation+rate+in+Gaddi+goat's+skin+fibroblast+cell+line.">Replicative senescence, telomere shortening and cell proliferation rate in Gaddi goat's skin fibroblast cell line.</a> Cell Biology International. 31 (10), 1257-1264 (2007).</li><li>Michaeli, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+telomere+length+correlates+with+extended+female+fertility.">Leukocyte telomere length correlates with extended female fertility.</a> Cells. 11 (3), 513 (2022).</li><li>Lesmana, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+reference+intervals+for+leukocyte+telomere+length+in+children:+the+method+matters.">Continuous reference intervals for leukocyte telomere length in children: the method matters.</a> Clinical Chemistry and Laboratory Medicine. 59 (7), 1279-1288 (2021).</li></ol>

The authors have no conflicts of interest.

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 &#181;g of genomic DNA without affecting the results. Several other kit components, such as the DIG-labeled molecular weight m...

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&#39; 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&#8242;-(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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell Line</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A2780 (Ovarian adenocarcinoma cell line)</td>
			<td>Received as a gift</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc XRS+ (for imaging and UV cross linking)</td>
			<td>Biorad</td>
			<td>Universal hood II (721BR14277)</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop (Epoch 2)</td>
			<td>Biotek</td>
			<td>EPOCH2</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TeloTool</td>
			<td>Version 1.3</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Molychem</td>
			<td>64-19-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>MP</td>
			<td>180720</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Gibco, ThermoFisher Scientific, USA</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM&#160;</td>
			<td>HyClone, Cytiva, USA</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediamine tetraacetic acid&#160;</td>
			<td>Molychem</td>
			<td>6381-92-6</td>
			<td></td>
		</tr>
		<tr>
			<td>HI FBS</td>
			<td>Gibco, ThermoFisher Scientific, USA</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Molychem</td>
			<td>76-47-01-0</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Molychem</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Molychem</td>
			<td>1310-73-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon membrane</td>
			<td>Sigma</td>
			<td>11209299001</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Gibco, ThermoFisher Scientific, USA</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Affymetrix</td>
			<td>151-21-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Gibco, ThermoFisher Scientific, USA</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>BIORAD</td>
			<td>77-86-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris HCl</td>
			<td>Sigma Aldrich</td>
			<td>1185-53-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman paper</td>
			<td>GE healthcare lifesciences</td>
			<td>1001-917</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 kb ladder</td>
			<td>NEB</td>
			<td>N3232S</td>
			<td></td>
		</tr>
		<tr>
			<td>20x SSC</td>
			<td>Invitrogen</td>
			<td>15557-036</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti DIG AP</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking solution 10x</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cutsmart Buffer</td>
			<td>NEB</td>
			<td>B6004</td>
			<td></td>
		</tr>
		<tr>
			<td>Detection buffer 10x</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dig easy hyb</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Digestion Buffer</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hinf 1</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hinf 1 (alternative to kit)</td>
			<td>NEB</td>
			<td>R0155T</td>
			<td></td>
		</tr>
		<tr>
			<td>Loading Dye</td>
			<td>BIOLABS</td>
			<td>N3231S</td>
			<td></td>
		</tr>
		<tr>
			<td>Maleic acid buffer 10x</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular marker</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Probe</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Rsa 1</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Rsa 1 (alternative to kit)</td>
			<td>NEB</td>
			<td>R0167L</td>
			<td></td>
		</tr>
		<tr>
			<td>Substrate</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
		<tr>
			<td>Wash buffer</td>
			<td>Telo TAGGG Telomere Length Assay kit</td>
			<td>12209136001</td>
			<td></td>
		</tr>
	</tbody>
</table>

optimization of performance parameters of the taggg telomere length assay

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.

Author Spotlight: Optimization of Performance Parameters of the TAGGG Telomere Length Assay  

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

The scope of our research involves measuring telomere length differences across various study groups. The questions we are trying to answer revolve around diseases of physiological conditions and how they may affect telomere length and vice versa. The technology is currently used to advance research in the field of telomere length biology are nanopore sequencing and more recently CRISPR Cas based measurement.Current experimental challenges are accurately measuring absolute telomere length and the changes in it. The same sample could give varying results when different methods of measuring telomere length are used. The research gap we address is the lack of a standard method to measure telomere length.Telomere restriction fragment analysis or TRF, is still the standard against which all new techniques are evaluated. Also, our optimized protocols helps in processing a higher number of samples with limited resources. Our findings will help advance research in the field of telomere length biologic by providing a reliable and relatively economical way to compare newer methods to the gold standard, which is TRF.Our laboratory will focus on the effects of small molecule inhibitors on telomere length. We will also study the impact that lifestyle diseases and telomere length have on on each other.

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, ...

Watch this Scientific Journal Video about Optimization of Performance Parameters of the TAGGG Telomere Length Assay at JoVE.com

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 ...

optimization-performance-parameters-taggg-telomere-length

Research

JoVE Journal

Cancer Research

1.5K vues.  SVKM's NMIMS Deemed-to-be-University. La portée de notre recherche consiste à mesurer les différences de longueur des télomères entre divers groupes d’étude. Les questions auxquelles nous essayons de répondre tournent autour des maladies des conditions physiologiques et de la façon dont elles peuvent affecter la longueur des télomères et vice versa. La technologie actuellement utilisée pour faire progresser la recherche dans le domaine de la biologie de la longueur des télomères sont le séquençage des nanopores e...