The authors acknowledge the support of the University of Pittsburgh Cancer Institute Biobehavioral Oncology shared facility that is supported in part by award P30CA047904.

1. Genomic DNA Extraction
<ol>
	<li>Perform a genomic DNA extraction from the cells (here, cell line BJ-hTERT) to be analyzed using a commercial DNA extraction kit, according to the manufacturer&#39;s instructions.</li>
	<li>Measure the DNA concentration with a spectrophotometer. If the DNA concentration is less than 100 &#181;g/mL, precipitate the DNA with ethanol and resuspend in a volume of TE buffer (10 mM Tris-Cl; 0.1 mM EDTA (Ethylenediaminetetraacetic Acid); pH 8.0), to ensure a DNA concentration of 100-600 &#181;g/mL).</li>
</ol>
2. DNA Integrity Assessment
NOTE: This portion of the protocol outlines a DNA integrity assessment using a gel electrophoresis system with a 11 cm x 14 cm gel. Other systems and gel sizes can also be used, but the voltage and DNA migration time may vary.
<ol>
	<li>Prepare a 1% agarose gel by combining 1 g of electrophoresis grade agarose in 100 mL of 1x TAE buffer (40 mM Tris-base; 20 mM acetic acid; 2 mM EDTA; pH 8.5) and microwave until the agarose is dissolved and the solution is clear. Cool the solution at room temperature until it reaches 50 &#176;C (approximately 15 - 20 min).</li>
	<li>While agarose solution is cooling, prepare gel casting tray by adding casting end gates to the casting tray. To prevent agarose leakage, cool the end gates to 4 &#176;C prior to attaching to the casting tray.</li>
	<li>Once the agarose solution has cooled to 50 &#176;C, add 10 &#181;L of ethidium bromide (10 mg/mL in dH2O) and pour the agarose solution into the gel casting tray. Add the comb to the casting tray.</li>
	<li>Allow the agarose solution to harden for 45 min at room temperature.</li>
	<li>Prepare DNA samples for electrophoresis by diluting 25 ng of genomic DNA using 1x TE buffer to a final volume of 9 &#181;L and add 1 &#181;L of 10x Loading Dye (0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol FF, 30% (v/v) glycerol). Mix well.</li>
	<li>Prepare the gel electrophoresis system for DNA migration by filling the electrophoresis system with 1x TAE buffer so that the buffer level is several millimeters above the gel. Load the wells of the gel with the entirety of the DNA samples. Add 5 &#181;L of a DNA ladder to a single well.</li>
	<li>Run the DNA migration at 100 V for approximately 2 h or until the bromophenol blue dye is approximately half way through the gel.</li>
	<li>Image the gel using an ultraviolet light transilluminator or equivalent. 
	NOTE: DNA samples with good integrity have high molecular weight bands with no smearing (that is due to sheared or broken DNA). If the DNA bands are smeared, they have low integrity and are not suitable for the Southern blot analysis, and the DNA must be re-isolated with fresh cells.</li>
</ol>
3. Genomic DNA Digestion
<ol>
	<li>Perform the digestion of the genomic DNA in a final volume of 20-40 &#181;L. 
	NOTE: Volumes greater than 40 &#181;L will overflow the wells of the agarose gel.
	<ol>
		<li>Combine 3 &#181;g of genomic DNA and the appropriate amount of 10x DNA digest buffer (supplied with the restriction enzymes) so that the final concentration is 1x, in a microtube. Add 1 &#181;L of RsaI restriction enzyme (10,000 U/mL) and 1 &#181;L of HinfI restriction enzyme (10,000 U/mL) to each tube. Adjust the final volume using sterile, deionized H2O. Mix well. Centrifuge briefly (10 - 15 s at 16,000 x g) to ensure that all components are in the bottom of the tube.</li>
	</ol>
	</li>
	<li>Incubate the digestions at 37 &#176;C for &#8805;16 h. After the digestion, store the digested DNA at 4 &#176;C or -20 &#176;C for no longer than 2 days if needed17.</li>
</ol>
4. Genomic DNA Gel Electrophoresis
NOTE: This portion of the protocol outlines a procedure for using a 20 cm x 25 cm gel electrophoresis system. Different gel electrophoresis systems can be used, but several variables may need to be modified including voltage, time of migration and amount of electrophoresis buffer added to the system in order to yield optimal results.
<ol>
	<li>Prepare 0.6% agarose solution by combining 2.25 g of gel electrophoresis grade agarose with 375 mL of electrophoresis buffer, either 1x TAE or 0.5x TBE (110 mM Tris base; 90 mM boric acid; 2.5 mM EDTA; pH 8.3). 
	NOTE: For telomeres less than 8,000 kB, 0.5x TBE is recommended whereas 1x TAE is recommended for telomeres between 8,000 kB and 20,000 kB1.</li>
	<li>Microwave the solution until the agarose has dissolved and the solution is clear. Allow the solution to cool for 15 - 20 min at room temperature or until the agarose reaches 50 &#176;C.</li>
	<li>While the agarose solution is cooling, prepare the gel electrophoresis system for gel casting as described in step 2.2. Pour the agarose gel solution into a gel casting tray. Place a comb into the gel. Allow the gel to harden for 45 min uncovered at room temperature.</li>
	<li>Prepare the digested DNA samples for electrophoresis. For a 40 uL reaction, add 0.4 &#181;L of 10x loading dye into each sample of digested DNA. Briefly centrifuge (10-15 s, 16,000 x g) to ensure that all of the solution is at the bottom of the tube.</li>
	<li>Prepare the gel electrophoresis system for DNA migration. Remove the end gates and comb from the gel. Fill the gel electrophoresis system with 1x TAE or 0.5x TBE to the fill line, ensuring that the gel is completely submerged in the buffer.</li>
	<li>Load the wells of the gel with the DNA samples such that there is a blank well between samples. Avoid causing bubbles or puncturing the gel. Add 10 &#181;L of a DNA ladder into wells on the opposite ends of the gel. 
	NOTE: The type of DNA ladder will depend on the length of the telomeres, which will vary from person to person and between cell sources.</li>
	<li>Run the gel electrophoresis at 150 V for 30 min to quickly move the DNA into the gel; then adjust the voltage to 57 V and run for 18-24 h.</li>
</ol>
5. Gel Fluorescent Imaging and Hybridization
<ol>
	<li>After 18-24 h, turn off the gel electrophoresis power source. Remove the gel casting tray with the gel from the electrophoresis buffer. Slice off the top right corner of the gel to serve as a reference for the orientation of the gel.</li>
	<li>Prepare a piece of filter paper by cutting the paper to be a slightly larger size than the gel. Place the filter paper on top of the gel in the casting tray and allow the paper to soak up excess solution on the gel. Carefully remove the air bubbles between the filter paper and the gel using a pipette as a roller to squeeze the bubbles out through the sides.</li>
	<li>Remove the gel from the casting tray by flipping the gel and filter paper over so that the paper is underneath the gel. Transfer the gel with the filter paper to a gel dryer and cover the gel with plastic wrap. Remove all air bubbles between the plastic wrap and gel using a pipette as a roller.</li>
	<li>Dry the gel for 2 h at 50 &#176;C.</li>
	<li>Once dried, remove the plastic wrap and transfer the gel and filter paper to a glass dish containing 250 mL of deionized water (sufficient to cover the gel). Carefully remove the filter paper and incubate the gel at room temperature for 15 min. The gel will be thin, firm and easy to manipulate without tearing.</li>
	<li>Prepare 5x SSC solution (750 mM NaCl; 75 mM sodium citrate) in deionized water.</li>
	<li>Lay the gel on top of a nylon mesh (12 in x 12 in). Roll the nylon mesh and gel together making sure that the gel is not in contact with itself. Place the mesh and gel into a hybridization tube (e.g., 12 in tube with 1.5 in diameter). 
	NOTE: When properly rolled up, the gel will be in contact with the nylon mesh on both sides.</li>
	<li>Combine 100 mL of 5x SSC and 12 &#181;L of green fluorescent nucleic acid stain, and place into the hybridization tube. Protect the solution from light and incubate for 30 min at 42 &#176;C in a hybridization oven with rotation.</li>
	<li>Remove the nylon mesh/gel from the hybridization tube, roll open and place flat into a glass dish containing 250 mL of deionized water, and gently remove nylon mesh. Image the gel using a phosphorimager. Use the fluorescent settings: 520 BP, 40 Blue and 488 nm. Set the photomultiplier tube (PMT) to 375, the focal plane to +3 mm and the sensitivity to normal. Save the image to a file.</li>
</ol>
6. Hybridization
<ol>
	<li>Prepare the radioactive telomere probe.
	<ol>
		<li>Combine 2 &#181;L (2 ng) of telomere probe (5- (CCC TAA)4 3&#39;), 2.5 &#181;L of 10x polynucleotide kinase reaction buffer, 3 &#181;L of &#947;32P-dATP (6,000 Ci/mmol), 4 &#181;L of T4 polynucleotide kinase and DNase free, sterile deionized water to a final volume of 20 &#181;L in a microcentrifuge tube. 
		Caution: Follow facilities&#39; radioactive safety and regulations when using radioactivity.</li>
	</ol>
	</li>
	<li>Briefly centrifuge (10-30 s at 16,000 x g) and incubate in water bath at 37 &#176;C for 1 h. Centrifuge for 10-30 s at 16,000 x g and incubate at 65 &#176;C for 20 min to stop the reaction.</li>
	<li>Using a G-25 chromatography microspin column, mix the contents of the column by vortexing, then centrifuge for 1 min at 700 x g at room temperature. Discard the filtrate.</li>
	<li>Load the radioactive probe solution into the G-25 chromatography column and centrifuge for 2 min at 700 x g. Save the filtrate. 
	NOTE: Retain the filtrate as this contains the radioactive telomere probe. The radioactive probe can be stored at 4 &#176;C if needed.</li>
	<li>Prepare 250 mL of denaturing solution composed of 1.5 M NaCl and 0.5 M NaOH in sterile deionized water.</li>
	<li>Place the gel into a glass dish containing 250 mL of denaturing solution and incubate for 15 min at room temperature. Remove the denaturing solution and replace with 250 mL of sterile deionized water. Incubate for 10 min on an orbital shaker (40 - 50 rpm) at room temperature.</li>
	<li>Prepare 250 mL of neutralization solution composed of 1.5 NaCl and 0.5 M Tris-hydrochloric acid, pH 8.0 in sterile deionized water.</li>
	<li>Remove the deionized water and replace with 250 mL neutralization solution and incubate for 15 min at room temperature.</li>
	<li>During the incubation in the neutralization solution, prepare 50 mL hybridization solution composed of 5x SSC, 5x Denhardt&#39;s solution (0.1% non-ionic synthetic polymer; 0.1% polyvinyl pyrrolidine; 0.1% bovine serum albumin), 10 mM disodium phosphate (Na2HPO4) and 1 mM sodium pyrophosphate tetrabasic decahydrate (Na4P2O7 . 10 H2O) in sterile deionized water.</li>
	<li>Roll the gel into nylon mesh and place into the hybridization tube (step 5.7). Add 20 mL of the hybridization solution and incubate in a hybridization oven at 42 &#176;C for 10 min with rotation.</li>
	<li>Remove the hybridization solution and replace with 20 mL of fresh hybridization solution. Add the entirety of the telomere probe and incubate in a hybridization oven at 42 &#176;C overnight with rotation.</li>
</ol>
7. Gel Washing, Phosphor Screen Exposure, and Radio Imaging
<ol>
	<li>Prepare 500 mL of 2x SSC solution (300 mM NaCl; 30 mM sodium citrate) in sterile deionized water.</li>
	<li>Prepare 250 mL of 0.1x SSC (15 mM NaCl; 1.5 mM sodium citrate) and 0.1% Sodium Dodecyl Sulfate (SDS) in deionized water.</li>
	<li>Remove the gel and mesh from the hybridization tube and place into a glass dish containing 250 mL of 2x SSC. Unroll and remove the nylon mesh from the dish. Place the glass dish onto an orbital shaker and incubate for 15 min at room temperature.</li>
	<li>Remove the 2x SSC and replace with 250 mL of 0.1x SSC and 0.1% SDS solution, and incubate for 15 min on an orbital shaker at room temperature.</li>
	<li>During the incubation, expose the Phosphor screen to the image eraser for 15 min.</li>
	<li>Remove the 0.1x SSC and 0.1% SDS solution and replace with 250 mL of 2x SSC. Incubate for 15 min on an orbital shaker.</li>
	<li>Remove the gel from 2x SSC solution and either wrap the gel in plastic wrap or place into a heat sealable pouch. Remove all bubbles and air pockets between the gel and the plastic wrap or plastic pouch. Place the gel into an exposure cassette with the Phopshor screen. Expose the Phosphor screen to the gel overnight.</li>
	<li>Image the exposed Phosphor screen using a phosphorimager.</li>
</ol>
8. Telomere Length Quantification
<ol>
	<li>Open the phosphorimager file containing the fluorescent image of the green fluorescent nucleic acid-stained gel (Figure 1).</li>
	<li>Select &#39;Tools&#39; and then &#39;Pixel Distance&#39;. Using the mouse, draw a line from the bottom of the gel well to the middle of the first marker band and record the distance. Repeat this procedure for each marker band (Figure 2). These distances will be used in the graphing program as described below.</li>
	<li>Open the phosphorimage of the radiolabeled gel (Figure 3). Select &#39;Object Manager&#39; and then &#39;Grid&#39;. Set the Grid to 1 column and 150 rows. Adjust the grid so that it stretches from the top of the gel to the bottom of the gel. Adjust the width of the grid to equal the width of the lane, clicking on the edge of the grid and dragging the width of the grid to equal the width of the lane.</li>
	<li>Copy this grid (so that the width and height of the boxes remain the same) and move the second grid to an empty lane (for background purposes). Continue to copy and move additional grids for additional sample lanes. 
	NOTE: When finished, there should be grids for each sample lane and one grid of an empty lane for background (Figure 4).</li>
	<li>Select &#39;Analysis&#34; and then &#39;Volume Report&#39;. Select all of the grids for the report. Once the volume report is displayed, double click on the report to activate the spreadsheet program. Save the spreadsheet file. The data for this file will be under the &#39;Volume&#39; lane.</li>
	<li>Open an appropriate graphing program and create a new data and table. Select &#39;Y: Enter and plot a single Y value for each point&#39; then select &#39;Create&#39;.</li>
	<li>Label the first column (Column X) as &#39;Measured Distance&#39; and label the second column (Column Y) as &#39;Molecular Weight&#39;. Enter the marker molecular weights of each marker band in column Y. Enter the migration distances obtained in step 8.2 corresponding to each molecular weight marker.</li>
	<li>Go to &#39;Analysis&#39; and select &#39;Analyze&#39; and then &#39;nonlinear regression (curve fit)&#39; and select &#39;OK&#39;. At the next screen, select &#39;Exponential and then &#39;One phase decay&#39;. Select &#39;Range&#39; tab and enter &#39;150&#39; in &#39;number of points that define the curve&#39; and check &#39;Create a table of XY coordinates to export or copy the curve to another program&#39;. 
	NOTE: The result, &#39;nonlinear fit of Data 1&#39;, contains the molecular weight for each distance in the Y row.</li>
	<li>For analysis of the sample lanes, select an area of analysis that is above and below the actual telomere bands. Do not use the entire length of the lane (Figure 5). Determine the grid locations for the area of analysis of each lane.</li>
	<li>Select &#39;File&#39;, &#39;New&#39; &#39;Create new Table and Graph&#39;. Select &#39;Y: Enter and plot a single Y value for each point&#39; then select &#39;Create&#39;. Copy and paste the background lane volume values (intensity values) from the spreadsheet file into the first column (X) and the volume values (intensity values) from the sample lane into the first column Y. If there are multiple sample lanes, copy each set of volume values from the spreadsheet file into additional Y columns in the graphing program file.</li>
	<li>Delete the values from the background column (X) and each sample lane (Y) that are outside the areas of analysis determined in step 8.8. 
	NOTE: The data table should only contain values in those grid locations corresponding to the areas of analysis.</li>
	<li>Select Analysis, then &#39;Analyze&#39; and &#39;transform&#39;. Click &#39;OK&#39;. Select &#39;Transform Y values using Y=Y-X&#39;. This will transform the data in Data 2 (transformed intensity value (Y) = sample intensity (Y) - background intensity (X)).
	<ol>
		<li>Select Analysis then &#39;Analyze&#39;, &#39;Column Analysis&#39; and &#39;Column statistics&#39;. Select OK. The results tab &#39;Col. Stats of Transform of Data 2&#39; shows under the &#39;Sum&#39; row, &#8721;(Inti).</li>
	</ol>
	</li>
	<li>Select &#39;File&#39;, &#39;New&#39;, &#39;Create New Table and Graph&#39;. Select &#39;Y: Enter and plot a single Y value for each point&#39; then select &#39;Create&#39;. Copy and paste the Y column data from the results page &#39;Nonlin fit of Data 1, Curve&#39; into the new X column. Copy and paste the Y column data from the results page &#39;Transform of Data 2&#39; into the new Y column. Select Analysis then &#39;Analyze&#39; and &#39;Transform&#39;. Click &#39;OK&#39;. Select &#39;Transform Y values using Y=Y/X&#39;.
	<ol>
		<li>Select Analysis then &#39;Analyze&#39;, &#39;Column Analysis&#39; and &#39;Column statistics&#39;. Select OK. The results tab &#39;Col. Stats of Transform of Data 3&#39; shows under the &#39;Sum&#39; row, &#8721;(Inti/MWi). MW=molecular weight.</li>
	</ol>
	</li>
	<li>To calculate the average telomere length (TL) for each sample, use the formula TL=&#8721;(Inti)/ &#8721;(Inti/MWi).</li>
</ol>

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The authors have nothing to disclose.

Following electrophoresis, the genomic DNA smear is higher than 800 bp. This can occur if the restriction enzymes are faulty or if additional enzymes (as described above) are needed. A second possible explanation is that the protein is still attached to the DNA. When determining the DNA concentration by spectrophotometer, the 260/280 ratio should be around 1.8. If this ratio is &#60;1.5, there is protein contamination which can interfere with restriction enzyme digestion. Either the DNA sample would need to be re-isolate...

Telomeres, situated at the ends of chromosomes, are nucleotide repeats of the sequence TTAGGG (on human chromosomes) that play a critical role in protecting cellular genetic information3,4,5. Telomeres are several thousand base pairs in length and serve to protect the integrity of the rest of the chromosome during DNA replication. Replication of chromosomes is not perfect resulting in the ends of the chromosome not being completely copied. This inefficiency in replication is termed the &#34;end-replication problem&#34; and results in loss of some of the telomere repeats during each round of replication6,7. Telomere length can be restored after cell division by telomerase, an enzyme that replaces the lost telomere sequences8,9. Telomerase, however, is not activated in most human somatic cells and as a result, over time, telomeres will shorten, eventually resulting in cellular senescence10. Due to telomere shortening, telomeres are regarded as a biomarker of aging and risk of age-related diseases11,12. Additionally, telomere length can be affected by genetic, environmental, and lifestyle factors and other stimuli such as oxidative stress, indicating that telomere length can play a role as a biomarker in toxicological, epidemiological and behavioral studies13,14,15.
This article demonstrates a technique for measuring telomere length using a modified terminal restriction fragment (TRF) analysis adapted from Mender and Shay2 and Kimura et al.16, and similar to Herbert, Shay and Wright1 and Haussmann and Mauck17. Traditionally, TRF analysis involves a Southern blot procedure. Southern blots are considered the &#34;gold-standard&#34; for studying telomere length and are actively used for examining leukocyte telomere length in epidemiology studies18. This TRF analysis uses digested genomic DNA leaving behind the telomere repeats. The DNA fragments are then separated by gel electrophoresis, denatured and transferred to a nitrocellulose membrane. The telomere sequences are hybridized to a telomere oligonucleotide probe. Rather than transferring the separated telomeres to a membrane, other TRF techniques use in-gel hybridization techniques with and without denaturation of the DNA. The inclusion of denaturation is important when considering the detection of interstitial telomeres, which have been reported in some species19. Procedures that lack denaturing steps only detect the single stranded DNA overhangs at terminal telomeres and will not bind to interstitial telomere sequences18.
Besides TRF analysis, there are several other techniques for measuring telomere length that each have their own advantages and disadvantages. The Flow-FISH technique can measure mean telomere length of individual cells of a distinct cell type using flow cytometry16,20. While it can accurately tag telomeres with highly specific probes and reduce human error through automation, Flow-FISH is expensive, less efficient and requires fresh samples and a highly-skilled technician, limiting its ability for epidemiology studies16. In addition, this method does not account for potential changes in the number of chromosomes in the cell population. Another technique, qPCR, is widely accepted as a means of measuring telomere length for epidemiology studies16 due to its high-throughput, low-cost and requirement for small amounts of DNA compared to other methods. However, qPCR can only measure average telomeric DNA content relative to a single copy gene and is, thus, an indirect estimate of average telomere length. It also yields a high coefficient of variance in comparison studies, compared to southern blots, leading to questionable accuracy21.
The TRF procedure has its own deficiencies that limit its use for some studies. TRF techniques require a large amount of DNA compared to other methods (between 2 and 3 &#181;g per sample). This limits the technique&#39;s applications to examining telomere length of clinical samples for which sufficient genomic DNA can be collected, and cannot be used on degraded DNA samples. Additionally, the TRF analysis is costly and labor intensive, requiring 4-5 days to yield results. However, the TRF yields a low coefficient of variance granting its reproducibility. While this protocol is different than the traditional Southern blot (utilizing in situ gel hybridization), the two techniques are fundamentally the same.
The technique presented here is a combination of a Southern blot and in-gel hybridization; associating the DNA denaturing step from Southern blots with the in-gel hybridization. This combination has the added benefit of improved probe signal strength compared to the Southern blot and has consistently yielded quantifiable results within our lab. Additionally, the use of radioactive probes rather than chemiluminescent probes yields higher signal intensity and allows for visualization and quantification with a phosphorimager, making the analyses of the TRF user-friendly.

<table><tbody><tr><td>&lt;strong&gt;Biologics&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Rsa1, restriction enzyme</td><td>New England Biolabs, Inc</td><td>R0167S</td><td>10,000 U/mL, DNA digestion.&amp;nbsp; Comes with Cutsmart Buffer (digestion buffer)</td></tr><tr><td>Hinf1, restriction enzyme</td><td>New England Biolabs, Inc</td><td>R0155S</td><td>10,000 U/mL, DNA digestion.&amp;nbsp; Comes with Cutsmart Buffer (digestion buffer)</td></tr><tr><td>Seakem GTG Agarose</td><td>Lonza</td><td>50071</td><td>electrophoresis grade agarose</td></tr><tr><td>Optikinase&amp;nbsp;</td><td>affymetrix</td><td>78334X 500 UN</td><td>T4 Polynucleotide Kinase</td></tr><tr><td>SYBR Green Nucleic Acid Stain I</td><td>ThermoFisher Scientific</td><td>S7567</td><td>Syber Green</td></tr><tr><td>exactGene DNA Ladder 24- kB</td><td>Fisher Scientific</td><td>BP2580100</td><td></td></tr><tr><td>C-Strand Probe (3&#039;-(G3AT2)4-&#039;5)</td><td>Integrated DNA Technologies</td><td></td><td>Custom ordered DNA oligonucleotide</td></tr><tr><td>Gamma-ATP-P32</td><td>Perkin Elmer</td><td>BLU502Z</td><td>Radioisotope</td></tr><tr><td>Qiagen Blood and Cell Culture DNA Mini Kit</td><td>Qiagen</td><td>13323</td><td>DNA extraction kit</td></tr><tr><td>GE Illustra Microspin G-25 column</td><td>GE Healthcare Life Sciences</td><td>27-5325-01</td><td>For purification of readioactive oligo probe</td></tr><tr><td>Ficoll 400</td><td></td><td></td><td>non-ionic synthetic polymer</td></tr><tr><td>&lt;strong&gt;Equipment/Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Owl A5 Gel electrophoresis system (20 cm x 25 cm)</td><td>ThermoFisher Scientific</td><td>A5</td><td>Gel electrophoresis</td></tr><tr><td>Horizon 11.4 Gel electrophoresis system (11 cm x 14 cm)</td><td>Corel Life Sciences</td><td>11068020</td><td>Gel electrophoresis</td></tr><tr><td>Nylon mesh, 50, 12&quot; x 12&quot;</td><td>Ted Pellam, Inc</td><td>41-12105</td><td></td></tr><tr><td>GE Storage Phosphor Screens</td><td>GE Healthcare Life Sciences</td><td>28-9564-75</td><td></td></tr><tr><td>Phosphor Screen Exposure Cassette</td><td>GE Healthcare Life Sciences</td><td>63-0035-44</td><td></td></tr><tr><td>Isotemp Hybridzation Incubator</td><td>Fisher Scientific</td><td>13-247-20Q</td><td></td></tr><tr><td>Cylindrical hybridization tubes</td><td>Fisher Scientific</td><td>13-247-300</td><td></td></tr><tr><td>The Belly Dancer orbital shaker</td><td>Sigma-aldrich</td><td>Z768499-1EA</td><td>Orbital shaker</td></tr><tr><td>Typhoon 9400</td><td>ABI</td><td></td><td>For imaging of gels and phosphor screens</td></tr><tr><td>GraphPad Prism 6</td><td>GraphPad</td><td>Version 6.05</td><td>Graphing Program</td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td>Office 365</td><td>Spreadsheet program</td></tr><tr><td>Nanodrop</td><td>Thermo Scientific</td><td>Nanodrp 2000</td><td>Spectrophotometer</td></tr></tbody></table>

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.

Three concentrations of DNA (1, 2 and 3 &#181;g) isolated from the cell line BJ-hTERT (telomerase immortalized human foreskin fibroblasts)22 and human peripheral blood mononuclear cells (PBMCs) were analyzed for telomere length. Table 1 shows the lane assignments for each DNA sample. Figure 1 shows a nucleic acid fluorescent stain of the gel. The molecular weight standards are clearly visible. The distance from the top...

Watch this Scientific Journal Video about Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization at JoVE.com

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

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 ...

modified-terminal-restriction-fragment-analysis-for-quantifying

Research

JoVE Journal

Cancer Research

12.8K Views.  University of Pittsburgh.  In dit artikel wordt een gedetailleerd protocol voor het kwantificeren van telomere lengte met behulp van een gewijzigde terminal restrictie fragment analyse besproken die snelle en efficiënte directe meting van telomere lengte biedt. Deze techniek kan toegepast worden op een verscheidenheid aan celbronnen van DNA voor het kwantificeren van telomere lengte. 

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

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate for telomerase for de novo telomere DNA synthesis and is therefore important for telomere maintenance. T. brucei is a protozoan parasite that causes sleeping sickness in humans and nagana in cattle. Once infected mammalian host, T. brucei cell regularly switches its surface antigen to evade the host's immune attack. We have recently demonstrated that the T. brucei telomere structure plays an essential role in regulation of surface antigen gene expression, which is critical for T. brucei pathogenesis. However, T. brucei telomere structure has not been extensively studied due to the limitation of methods for analysis of this specialized structure. We have now successfully adopted the native in-gel hybridization and ligation-mediated primer extension methods for examination of the telomere G-overhang structure and an adaptor ligation method for determination of the telomere terminal nucleotide in T. brucei cells. Here, we will describe the protocols in detail and compare their different advantages and limitations.

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

Telomeres are essential for chromosome stability and the telomere G-overhang structure is essential for telomerase-mediated telomere maintenance. We have recently adopted two methods for detecting the telomere G-overhang structure in Trypanosoma brucei, which are native in-gel hybridization and ligation-mediated primer extension, which will be described.

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate ...

Immunology and Infection

Laparoscopic Pancreatoduodenectomy With Modified Blumgart Pancreaticojejunostomy

Minimally invasive pancreatic resections are technically demanding but rapidly increasing in popularity. In contrast to laparoscopic distal pancreatectomy, laparoscopic pancreatoduodenectomy (LPD) has not yet obtained wide acceptance, probably due to technical challenges, especially regarding the pancreatic anastomosis.
The study describes and demonstrates all steps of LPD, including the modified Blumgart pancreaticojejunostomy. Indications for LPD are all pancreatic and peri-ampullary tumors without vascular involvement. Relative contra-indications are body mass index &#62;35 kg/m2, chronic pancreatitis, mid-cholangiocarcinomas and large duodenal cancers.
The patient is in French position, 6 trocars are placed, and dissection is performed using an (articulating) sealing device. A modified Blumgart end-to-side pancreaticojejunostomy is performed with 4 large needles (3/0) barbed trans-pancreatic sutures and 4 to 6 duct-to-mucosa sutures using 5/0 absorbable multifilament combined with a 12 cm, 6 or 8 Fr internal stent using 3D laparoscopy. Two surgical drains are placed alongside the pancreaticojejunostomy.
The described technique for LPD including a modified Blumgart pancreatico-jejunostomy is well standardized, and its merits are currently studied in the randomized controlled multicenter trial. This complex operation should be performed at high-volume centers where surgeons have extensive experience in both open pancreatic surgery and advanced laparoscopic gastro-intestinal surgery.

Laparoscopic pancreatoduodenctomy (LPD) may offer advantages over open pancreatoduodenectomy, including early postoperative mobilization, less delayed gastric emptying and a shorter hospital stay. However, LPD is technically challenging and not well-standardized, especially regarding the pancreatic anastomosis. We describe a standardized technique for the pancreatic anastomosis during LPD: modified Blumgart pancreaticojejunostomy.

Minimally invasive pancreatic resections are technically demanding but rapidly increasing in popularity. In contrast to laparoscopic distal ...

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. Recent findings revealed that TERRA molecules also interact with internal chromosomal regions to regulate gene expression in mouse embryonic stem (ES) cells. In line with this evidence, RNA fluorescence in situ hybridization (RNA-FISH) analyses have shown that only a subset of TERRA transcripts localize at chromosome ends. A better understanding of the dynamics of TERRA molecules will help define their function and mechanisms of action. Here, we describe a method to label and visualize single-telomere TERRA transcripts in cancer cells using the MS2-GFP system. To this aim, we present a protocol to generate stable clones, using the AGS human stomach cancer cell line, containing MS2 sequences integrated at a single subtelomere. Transcription of TERRA from the MS2-tagged telomere results in the expression of MS2-tagged TERRA molecules that are visualized by live-cell fluorescence microscopy upon co-expression of a MS2 RNA-binding protein fused to GFP (MS2-GFP). This approach enables researchers to study the dynamics of single-telomere TERRA molecules in cancer cells, and it can be applied to other cell lines.

Here, we present a protocol to generate cancer cell clones containing a MS2 sequence tag at a single subtelomere. This approach, relying on the MS2-GFP system, enables visualization of the endogenous transcripts of telomeric repeat-containing RNA (TERRA) expressed from a single telomere in living cells.

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in ...

Genetics

Droplet Digital TRAP (ddTRAP): Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

The telomere repeat amplification protocol (TRAP) is the most widely used assay to detect telomerase activity within a given a sample. The polymerase chain reaction (PCR)-based method allows for robust measurements of enzyme activity from most cell lysates. The gel-based TRAP with fluorescently labeled primers limits sample throughput, and the ability to detect differences in samples is restricted to two fold or greater changes in enzyme activity. The droplet digital TRAP, ddTRAP, is a highly sensitive approach that has been modified from the traditional TRAP assay, enabling the user to perform a robust analysis on 96 samples per run and obtain absolute quantification of the DNA (telomerase extension products) input within each PCR. Therefore, the newly developed ddTRAP assay overcomes the limitations of the traditional gel-based TRAP assay and provides a more efficient, accurate, and quantitative approach to measuring telomerase activity within laboratory and clinical settings.

Droplet Digital TRAP ddTRAP: Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

We successfully converted the standard telomere repeat amplification protocol (TRAP) assay to be employed in droplet digital polymerase chain reactions. This new assay, called ddTRAP, is more sensitive and quantitative, allowing for better detection and statistical analysis of telomerase activity within various human cells.

The telomere repeat amplification protocol (TRAP) is the most widely used assay to detect telomerase activity within a given a sample. The polymerase ...

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. ...

Quantifying Telomere Length Using Non-Radioactive Chemiluminescence Detection

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  

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 ...

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Studying TRF1/2 Interactions with Telomeric DNA Using Single-Molecule Assay

Analyzing Telomeric Protein-DNA Interactions Using Single-Molecule Magnetic Tweezers

Telomeres, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper function depends on tightly regulated processes of replication, elongation, and damage response. The shelterin complex, especially Telomere Repeat-binding Factor 1 (TRF1) and TRF2, plays a pivotal role in telomere protection and has emerged as a potential anti-cancer target for drug discovery. These proteins bind to the repetitive telomeric DNA motif TTAGGG, facilitating the formation of protective structures and recruitment of other telomeric proteins. Structural methods and advanced imaging techniques have provided insights into telomeric protein-DNA interactions, but probing the dynamic processes requires single-molecule approaches. Tools like magnetic tweezers, optical tweezers, and atomic force microscopy (AFM) have been employed to study telomeric protein-DNA interactions, revealing important details such as TRF2-dependent DNA distortion and telomerase catalysis. However, the preparation of single-molecule constructs with telomeric repetitive motifs continues to be a challenging task, potentially limiting the breadth of studies utilizing single-molecule mechanical methods. To address this, we developed a method to study interactions using full-length human telomeric DNA with magnetic tweezers. This protocol describes how to express and purify TRF2, prepare telomeric DNA, set up single-molecule mechanical assays, and analyze data. This detailed guide will benefit researchers in telomere biology and telomere-targeted drug discovery.

Author Spotlight: Advanced Single-Molecule Techniques for Investigating Telomeric Protein-DNA Interactions

This protocol demonstrates using single-molecule magnetic tweezers to study interactions between telomeric DNA-binding proteins (Telomere Repeat-binding Factor 1 [TRF1] and TRF2) and long telomeres extracted from human cells. It describes the preparatory steps for telomeres and telomeric repeat-binding factors, the execution of single-molecule experiments, and the data collection and analysis methods.

Telomeres, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper ...

Biochemistry

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 ...

Biology

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

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

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