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 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.

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><tbody><tr><td>&lt;strong&gt;Cell Line&lt;/strong&gt;</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>&lt;strong&gt;Equipment&lt;/strong&gt;</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>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>TeloTool</td><td>Version 1.3</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</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&amp;nbsp;</td><td>HyClone, Cytiva, USA</td><td>SH30243.01</td><td></td></tr><tr><td>Ethylenediamine tetraacetic acid&amp;nbsp;</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>&lt;strong&gt;Reagents&lt;/strong&gt;</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.

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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65288/65288fig01.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65288/65288fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65288/65288fig02.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65288/65288fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65288/65288fig03.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65288/65288fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65288/65288_Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65288/65288_Table 2.xlsx" target="_blank">Please click here to download this Table.</a>

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

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

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.

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 ...

optimization-performance-parameters-taggg-telomere-length

Nanyang Technological University

Research

JoVE Journal

Cancer Research

2.7K Views.  SVKM's NMIMS Deemed-to-be-University. De reikwijdte van ons onderzoek omvat het meten van telomeerlengteverschillen tussen verschillende studiegroepen. De vragen die we proberen te beantwoorden, draaien om ziekten van fysiologische aandoeningen en hoe deze de telomeerlengte kunnen beïnvloeden en vice versa. De technologie wordt momenteel gebruikt om onderzoek te bevorderen op het gebied van telomeerlengtebiologie, nanoporiënsequencing en meer recent crispr cas-gebaseerde metingen.De huidige experimentele uitdagingen zijn...

Video: Auteur in de schijnwerpers: optimalisatie van de prestatieparameters van de TAGGG-telomeerlengtetest  

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy. Using this approach, researchers can accurately evaluate novel nanoscale formulations in relevant mouse models of cancer. These protocols may have the ability to identify the most promising cancer nanomedicines with high translational potential or to aid in the optimization of cancer nanomedicines for future translation.

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization

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.

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 ...

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an &#34;in-incubator&#34; video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.

This study reports two different methods for the analysis of cell invasion and migration: the Boyden chamber assay and the in vitro video microscope-based wound-healing assay. The protocols for these two experiments are described, and their benefits and disadvantages are compared.

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

Using the Dot Assay to Analyze Migration of Cell Sheets

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Cell migration is essential for development, tissue maintenance and repair, and tumorigenesis, and is regulated by growth factors, chemokines, and cytokines. This protocol describes the dot assay, a two-dimensional, unconstrained migration assay to assess the migratory phenotype of attached, cohesive cell sheets in response to microenvironmental cues.

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The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

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A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

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Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay

Estrogen receptor alpha (ER&#945;) is an estrogenic ligand-dependent transcription regulator. The sequence of ER&#945; protein is highly conserved among species. It has been thought that the function of human and mouse ER&#945;s is identical. We demonstrate the differential 4-hydroxy-tamoxifen (4OHT) effect on mouse and human ER&#945; ligand-binding domain (LBD) dimerization activity using the mammalian two-hybrid (M2H) assay. The M2H assay can demonstrate the efficiency of LBD homodimerization activity in mammalian cells, utilizing the transfection of two protein expression plasmids (GAL4 DNA-binding domain [DBD] fusion LBD and VP16 transactivation domain [VP16AD] fusion LBD) and a GAL4-responsive element (GAL4RE) fused luciferase reporter plasmid. When the GAL4DBD fusion LBD and the VP16AD fusion LBD make a dimer in the cells, this protein complex binds to the GAL4RE and, then, activates a luciferase gene expression through the VP16AD dependent transcription activity. The 4OHT-mediated luciferase activation is higher in the HepG2 cells that were transfected with the mouse ER&#945; LBD fusion protein expression plasmids than in the human ER&#945; LBD fusion protein expression plasmid transfected cells. This result suggests that the efficacy of the 4OHT-dependent mouse ER&#945; LBD homodimerization activity is higher than human ER&#945; LBD. In general, the utilization of the M2H assay is not ideal for the evaluation of nuclear receptor LBD dimerization activity, because agonistic ligands enhance the basal level of the LBD activity and that impedes the detection of LBD dimerization activity. We found that 4OHT does not enhance ER&#945; LBD basal activity. That is a key factor for being able to determine and detect the 4OHT-dependent LBD dimerization activity for successfully using the M2H assay. ER&#945; LBD-based M2H assays may be applied to study the partial agonist activity of selective estrogen receptor modulators (e.g., 4OHT) in various mammalian cell types.

We present a method for analyzing the 4-hydroxy-tamoxifen-dependent estrogen receptor alpha ligand-binding domain dimerization activity using the mammalian two-hybrid assay.

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Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

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Accurate Telomere Length Assessment in Human Population Studies

Monochrome Multiplex Quantitative PCR Telomere Length Measurement

Telomeres are ribonucleoprotein structures at the end of all eukaryotic chromosomes that protect DNA from damage and preserve chromosome stability. Telomere length (TL) has been associated with various exposures, biological processes, and health outcomes. This article describes the monochrome multiplex quantitative polymerase chain reaction (MMqPCR) assay protocol routinely conducted in our laboratory for measuring relative mean TL from human DNA. There are several different PCR-based TL measurement methods, but the specific protocol for the MMqPCR method presented in this publication is repeatable, efficient, cost-effective, and suitable&#160;for population-based studies. This detailed protocol outlines all information necessary for investigators to establish this assay in their laboratory. In addition, this protocol provides specific steps to increase the reproducibility of TL measurement by this assay, defined by the intraclass correlation coefficient (ICC) across repeated measurements of the same sample. The ICC is a critical factor in evaluating expected power for a specific study population; as such, reporting cohort-specific ICCs for any TL assay is a necessary step to enhance the overall rigor of population-based studies of TL. Example results utilizing DNA samples extracted from peripheral blood mononuclear cells demonstrate the feasibility of generating highly repeatable TL data using this MMqPCR protocol.

Author Spotlight: Exploring the Impact of Trauma on Cellular Aging

Here we present a protocol for the measurement of relative telomere length (TL) using the monochrome multiplex quantitative polymerase chain reaction (MMqPCR) assay. The MMqPCR assay is a repeatable, efficient, and cost-effective method for measuring TL from human DNA in population-based studies.

Telomeres are ribonucleoprotein structures at the end of all eukaryotic chromosomes that protect DNA from damage and preserve chromosome stability. ...

Genetics

Telomere Length and Telomerase Activity; A Yin and Yang of Cell Senescence

Telomeres are repeating DNA sequences at the tip ends of the chromosomes that are diverse in length and in humans can reach a length of 15,000 base pairs. The telomere serves as a bioprotective mechanism of chromosome attrition at each cell division. At a certain length, telomeres become too short to allow replication, a process that may lead to chromosome instability or cell death. Telomere length is regulated by two opposing mechanisms: attrition and elongation. Attrition occurs as each cell divides. In contrast, elongation is partially modulated by the enzyme telomerase, which adds repeating sequences to the ends of the chromosomes. In this way, telomerase could possibly reverse an aging mechanism and rejuvenates cell viability. These are crucial elements in maintaining cell life and are used to assess cellular aging. In this manuscript we will describe an accurate, short, sophisticated and cheap method to assess telomere length in multiple tissues and species. This method takes advantage of two key elements, the tandem repeat of the telomere sequence and the sensitivity of the qRT-PCR to detect differential copy numbers of tested samples. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

An accurate, short, sophisticated and cheap method is described that assesses telomere length in multiple tissues and species using qRT-PCR. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

Telomeres  are repeating DNA sequences at the tip ends of the chromosomes that are diverse  in length and in humans can reach a length of 15,000 base ...

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

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