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 (DMEM) complete medium supplemented with 10% fetal bovine serum (FBS), streptomycin, penicillin, and amphotericin B in a 6 cm Petri dish. Incubate at 37 &#176;C in a humidified and controlled environment containing 5% carbon dioxide until the cells are 80%-100% confluent.</li>
	<li>Remove the media and wash with 5 mL of 1x phosphate-buffered saline (PBS).</li>
	<li>Treat the cells with 1 mL of trypsin-ethylenediaminetetraacetic acid (EDTA) for detaching the cells by incubating at 37 &#176;C for 3-5 min.</li>
	<li>Add 2 mL of DMEM complete media to inactivate the trypsin and collect the cells in a centrifugation tube.</li>
	<li>Pellet the cells at 2,348 &#215; g for 5 min.</li>
	<li>Wash the pellet with 1x PBS and centrifuge at 2,348 &#215; g for 5 min.</li>
	<li>Store the pellet at -80 &#176;C until further use. 
	&#8203;NOTE: The cell count can vary depending on the cell line. We obtained approximately 2.5 &#215; 106 cells in the case of the A2780 cell line, which is used in further steps.</li>
</ol>
2. Genomic DNA isolation
<ol>
	<li>Add 500 &#181;L of lysis buffer (10 mM tris-Cl, pH 8.0; 25 mM EDTA, pH 8.0; 100 mM NaCl; 0.5% w/v sodium dodecyl sulphate) to the cell pellet and gently mix using a cut tip (with an opening diameter of a minimum 2 mm). Add 20 &#181;g/mL freshly prepared RNase A and mix gently by inversion.</li>
	<li>Incubate at 37 &#176;C for 30 min and occasionally invert the tube during incubation.</li>
	<li>Add proteinase K to a final concentration of 100 &#181;g/mL and mix gently by inversion 10 times. Incubate at 55 &#176;C for 2 h. Invert the tube at regular intervals (every 10 min) during incubation.</li>
	<li>Add 500 &#181;L of phenol:chloroform:isoamyl alcohol reagent (25:24:1) and mix gently by inversion 20 times. Centrifuge at 9,391 &#215; g for 15 min at room temperature (around 25 &#176;C). 
	NOTE: Figure 1A shows the three layers obtained after centrifugation.</li>
	<li>Remove the viscous upper aqueous layer out of the three layers visible, place into a fresh tube using a cut tip, and add an equal amount of chloroform. Mix by gentle inversion 20 times.</li>
	<li>Centrifuge the tubes at 9,391 &#215; g for 15 min at room temperature.</li>
	<li>Collect the aqueous layer and add an appropriate amount of 5 M NaCl so that the final concentration of NaCl is 0.2 M.</li>
	<li>Add two volumes of 100% ethanol. Mix by gentle inversion 20-25 times. Centrifuge at 15,871 &#215; g for 5 min at room temperature.</li>
	<li>Remove the supernatant and add 500 &#181;L of 70% ethanol to wash the pellet. Centrifuge at 15,871 &#215; g for 5 min at room temperature.</li>
	<li>Remove the supernatant, air-dry the pellet for a few minutes, and add 50 &#181;L of sterile nuclease-free water. Allow the DNA to rehydrate for 1-2 days at room temperature. Mix by pipetting, using the cut tip.</li>
	<li>Measure the concentration of DNA using ultraviolet (UV)-spectrophotometry. Redilute the samples, if necessary, using sterile nuclease-free water to get a minimum concentration of 300-500 ng/&#181;L.</li>
	<li>Check for the integrity of the DNA by running it on a 1% agarose gel (Figure 1B).</li>
	<li>Store the diluted DNA at -20 &#176;C until further use.</li>
</ol>
3. Digestion of genomic DNA
<ol>
	<li>Prepare an enzyme mixture of Rsa1 (20 U) and Hinf1 (20 U) and add 2 &#181;L of restriction digestion buffer per reaction.</li>
	<li>Take an appropriate volume of genomic DNA such that the total amount of DNA is 1.5 &#181;g. Make up the volume with sterile nuclease-free water, so that the total volume after the enzyme addition is 20 &#181;L.</li>
	<li>Mix well by tapping, followed by a pulse spin.</li>
	<li>Incubate the mixture at 37 &#176;C for 2 h.</li>
</ol>
4. Agarose gel electrophoresis
<ol>
	<li>Prepare a 10 cm &#215; 15 cm 0.8% agarose gel in 1x tris acetate EDTA (TAE) buffer using high-grade agarose.</li>
	<li>Add 5 &#181;L of loading dye to each digested genomic DNA sample, thus making the final volume 25 &#181;L, and load the samples onto the gel.</li>
	<li>Prepare a molecular marker mix by using 1 &#181;L of molecular marker (ladder), 3 &#181;L of sterile nuclease-free water, and 1 &#181;L of 5x loading dye.</li>
	<li>Load 5 &#181;L of molecular marker mix on both sides of the genomic DNA digested samples if there are a higher number of samples (~10), or only on one side if there are less than or equal to five samples.</li>
	<li>Run the gel at 5 V/cm for 6 h. Once the run is complete, make a notch on one corner of the gel to mark the loading order.</li>
	<li>Submerge the gel in 0.25 M HCl for 10 min at room temperature with gentle agitation.</li>
	<li>Rinse the gel twice with distilled water.</li>
	<li>Denature the DNA by submerging the gel in a solution of 0.5 M NaOH and 1.5 M NaCl twice for 15 min each at room temperature with gentle agitation.</li>
	<li>Rinse the gel twice with distilled water.</li>
	<li>Neutralize the DNA by submerging the gel in a solution of 0.5 M tris-HCl and 3 M NaCl (pH 7.5) twice for 15 min at room temperature with gentle agitation.</li>
</ol>
5. Southern blotting
<ol>
	<li>Cut a nylon membrane of 10 cm &#215; 12 cm in size and make a notch in the same position as the gel.</li>
	<li>Activate the membrane by dipping in distilled water followed by 20x sodium saline citrate (SSC) buffer (see Table 1).</li>
	<li>Set up the transfer.
	<ol>
		<li>Take a clean glass tray and place another tray in it in an inverted position. Create a wick using regular filter paper such that the ends are touching the base of the outer tray.</li>
		<li>Place the gel on top of the filter paper. Gently place the activated nylon membrane over the gel, with the notches overlapping, and remove any air bubbles by rolling over a glass rod.</li>
		<li>Place a 2 cm heap of 9.5 cm &#215; 11.5 cm cellulose filter paper, followed by a 6 cm heap of regular filter paper of the same dimensions. Place a weight atop this setup such that there is equal weight distribution below. Fill the outer tray with 20x SSC buffer (see Figure 2).</li>
		<li>Leave the setup for overnight transfer.</li>
	</ol>
	</li>
	<li>After the Southern transfer, fix the transferred DNA onto the membrane by UV crosslinking on a UV transilluminator or UV crosslinker. Use a 302 nm 8 W lamp for 5 min, or a 254 nm 8 W lamp for 2 min, which corresponds to approximately 120 mJ. Ensure that the membrane side on which the DNA is transferred faces the lamp.</li>
	<li>Wash the membrane twice with 25 mL of 2x SSC buffer.</li>
	<li>Pause the protocol, if required, by completely drying the blot and storing it at 2-8 &#176;C after loosely wrapping in foil, till further processing.</li>
</ol>
6. Hybridization and chemiluminescence detection
<ol>
	<li>Prewarm the prehybridization buffer to 42 &#176;C. 
	NOTE: The following steps include volumes of solutions/buffers to be used per 100 cm2 of blot.</li>
	<li>Incubate the membrane in 10 mL of prehybridization buffer for 1 h at 42 &#176;C with gentle agitation for prehybridization.</li>
	<li>Add 0.5 &#181;L of telomere probe per 5 mL of prewarmed prehybridization buffer to make the hybridization solution.</li>
	<li>Incubate the blot in 10 mL of hybridization solution at 42 &#176;C for 3 h with gentle agitation.</li>
	<li>Wash the blot with stringent buffer 1 (Table 1) twice for 10 min (25 mL each) at room temperature with gentle agitation.</li>
	<li>Prewarm stringent buffer 2 (Table 1) for 30 min at 50 &#176;C.</li>
	<li>Wash the blot with stringent buffer 2 twice for 15 min (25 mL each) at 50 &#176;C with gentle agitation.</li>
	<li>Rinse with 15 mL of wash buffer for 5 min with gentle agitation at RT.</li>
	<li>Incubate the membrane in 10 mL of freshly prepared 1x blocking solution for 30 min at room temperature with gentle agitation.</li>
	<li>Incubate the membrane in 10 mL of anti-digioxigenin conjugated with alkaline phosphatase working solution (see Table 2) for 30 min at RT with gentle agitation.</li>
	<li>Wash the blot twice with wash buffer at room temperature for 15 min with gentle agitation.</li>
	<li>Incubate in 10 mL of detection buffer for 5 min at RT with gentle agitation.</li>
	<li>Remove the excess detection buffer and keep the membrane with the DNA facing upward on a hybridization bag or acetate sheet. Add ~1-1.5 mL of substrate solution dropwise onto the membrane, immediately place another sheet on it, and incubate for 5 min at RT.</li>
	<li>Squeeze out the excess substrate solution for imaging.</li>
	<li>Image the blot for approximately 20 min in a gel documentation imaging system, collecting multiple images at different time points. Select unsaturated images for further analysis. 
	&#8203;NOTE: In case the signal is weak, imaging can be carried out for a longer period of time.</li>
</ol>
7. Analysis
<ol>
	<li>After obtaining the image files, export them for publication in .tif format at the highest resolution possible (600 dpi in this case).</li>
	<li>Install Telotool software. This requires a specific version (R2012a) of MATLAB (freely available) to run.</li>
	<li>Open the software and click on the Load Image File button in the top right.</li>
	<li>After the image has been loaded, click Invert Image, so that the image background is black and the smears/bands are white.</li>
	<li>Crop the image with the top corners starting from the wells. After the crop selection has been made, right-click inside it and select Crop Image.</li>
	<li>After the image has been cropped, click on Calculate Lanes and allow lane detection to occur automatically.</li>
	<li>If the lanes have not been detected properly, for example, if certain lanes have not been detected at all, or if extra or partial lanes have been detected, perform lane adjustment as described below.
	<ol>
		<li>Click on Adjust Lanes and in the pop-up window that opens, add and/or adjust lanes as needed, and click on Apply and Close.</li>
	</ol>
	</li>
	<li>After adjusting the lanes, make sure that a red dot is seen on each of the lanes and adjust the ladders using the Ladder Fit button, making sure that the number of peaks in both the ladder lanes are in the same position. Remove any extraneous peaks using the Delete Extremum button.</li>
	<li>After the ladders have been adjusted, select Lane Profiles, click on Ladder Fit, and select Polynomial Fit.</li>
	<li>Click on Trendline, as recommended by the software, and then select Corrected in the next option.</li>
	<li>Click on Get Results to display the results as a table, in which the Mean TRF row is the telomere length measurement of the respective lane samples.</li>
	<li>Click on Save all to store the results in the form of a spreadsheet.</li>
</ol>

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

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A2780 Cells Genomic DNA Isolation and Analysis

Begin by adding 500 microliters of the lysis buffer to the previously obtained A2780 cell pellet, and mix gently using a cut tip with a minimum opening diameter of two millimeters. Add 20 micrograms per milliliter of freshly prepared RNase and mix by gentle inversion. Incubate it at 37 degrees Celsius for 30 minutes.At the end of incubation, add proteinase K to a final concentration of 100 micrograms per milliliter and gently invert 10 times. Incubate again at 55 degrees Celsius for one hour inverting the tube every 10 minutes. Pipette 500 microliters of phenol:chloroform:isoamyl alcohol reagent into the tube and gently invert the tube about 20 times.Centrifuge it at 9, 391 G for 15 minutes at room temperature. Pipette the upper aqueous viscous layer into a new tube. Add an equal volume of chloroform and mix by gentle inversion.After centrifuging the tube once, collect the aqueous layer. Add an appropriate amount of five molar sodium chloride in the tube to increase the concentration by 0.2 moles. Add two volumes of 100%ethanol into the tube and invert it gently 20 times.Centrifuge at 15, 871 G for five minutes at room temperature. After discarding the supernatant, add 500 microliters of 70%ethanol. Centrifuge again at the same condition and then discard the supernatant.Once the pellet is air dried, add 50 microliters of sterile nuclease free water and let it rehydrate. Then mix the DNA by pipetting using the cut tip. Use UV spectrophotometry to measure the concentration of DNA.After digesting the DNA, separate it using 0.8%agarose. Submerge the agarose gel in 0.25 molar hydrochloric acid solution for 10 minutes at room temperature with gentle agitation. Then rinse the gel twice with distilled water.To denature the DNA, submerge the gel in a solution of sodium hydroxide and sodium chloride. Then rinse the gel twice with distilled water. To neutralize the DNA as demonstrated, submerge the gel in a solution of 0.5 molar hydrochloric acid and three molar sodium chloride at pH seven.The extractor genomic DNA showed good integrity indicating its use for further downstream processing of telomere restriction fragments.

Southern Blotting, Hybridization, and Chemiluminescence Detection of Genomic DNA Isolated from A2780 Cells

To perform southern blotting, start by cutting a nylon membrane into a 10 by 12 centimeter sized segment. Make a notch in the same position as the gel. Activate the membrane by dipping it in distilled water, followed by 20X sodium saline citrate buffer.Place a filter paper wick on a double inverted tray, set such that the ends touch the base of the outer tray. Place the gel over the filter paper. Then place the activated nylon membrane over the gel, ensuring that the notches overlap.Remove any air bubbles with a glass rod. Place a two centimeter heap of 9.5 by 11.5 centimeter sized cellulose filter paper, and another six centimeter heap of regular filter paper. Place a weight atop the setup for equal weight distribution.Fill the outer tray with 20X sodium saline citrate buffer, and leave it overnight for efficient transfer. After southern transfer, fix the transferred DNA on the membrane by UV crosslinking on a UV transilluminator. Wash the membrane twice with 25 milliliters of 2X sodium saline citrate buffer.To perform hybridization, incubate the membrane in 10 milliliters of the pre-warmed pre-hybridization buffer. Gently agitate the membrane manually at frequent intervals. Then incubate the blot in 10 milliliters of hybridization buffer at 42 degrees Celsius for three hours with gentle agitation.Wash the blot twice in 25 milliliters of stringent buffer for 10 minutes at room temperature with gentle agitation. Now wash the blot twice in 25 milliliters of pre-warmed stringent buffer at 50 degrees Celsius for 15 minutes with gentle agitation. Rinse with 15 milliliters of wash buffer for five minutes with gentle agitation at room temperature.Incubate the membrane in 10 milliliters of freshly prepared blocking solution for 30 minutes at room temperature with gentle agitation. Then place it in 10 milliliters of anti-digoxigenin conjugated with an alkaline phosphatase working solution. Wash the blot twice in the wash buffer at room temperature for 15 minutes.Now incubate in 10 milliliters of detection buffer for five minutes at room temperature. After removing the excess buffer, place the membrane on an acetate sheet with the DNA side facing up. Add approximately one to 1.5 milliliters of substrate solution dropwise on the membrane.Immediately place another acetate sheet on it and incubate for five minutes at room temperature. Squeeze out the excess substrate solution before imaging. Using a gel documentation imaging system, collect multiple unsaturated images at different time points for analysis.After southern blotting and hybridization, the telomere restriction fragments were clearly visible, indicated by a smear.

1.5K Views.  SVKM's NMIMS Deemed-to-be-University.  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. 

Quantifying Telomere Length Using Non-Radioactive Chemiluminescence Detection

Summary