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 &#18...

<ol>
	<li>Herbert, B. S., Shay, J. W., Wright, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+telomeres+and+telomerase.">Analysis of telomeres and telomerase.</a> Curr Protoc Cell Biol. , 16 (2003).</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 Protoc. 5 (22), (2015).</li><li>Blackburn, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeres+and+Telomerase:+The+Means+to+the+End+(Nobel+lecture).">Telomeres and Telomerase: The Means to the End (Nobel lecture).</a> Angew Chem Int Ed Engl. 49 (41), 7405-7421 (2010).</li><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=Telomerase+Discovery:+The+Excitement+of+Putting+Together+Pieces+of+the+Puzzle+(Nobel+lecture).">Telomerase Discovery: The Excitement of Putting Together Pieces of the Puzzle (Nobel lecture).</a> Angew Chem Int Ed Engl. 49 (41), 7422-7439 (2010).</li><li>Szostak, 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=DNA+Ends:+Just+the+Beginning+(Nobel+lecture).">DNA Ends: Just the Beginning (Nobel lecture).</a> Angew Chem Int Ed Engl. 49 (41), 7386-7404 (2010).</li><li>Watson, J. M., 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=Telomeres,+Aging,+and+Plants:+From+Weeds+to+Methuselah+-+A+Mini-Review.">Telomeres, Aging, and Plants: From Weeds to Methuselah - A Mini-Review.</a> Gerontology. 57 (2), 129-136 (2011).</li><li>Lu, W., Zhang, Y., Liu, D., Songyang, Z., Wan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeres-Structure,+Function,+and+Regulation.">Telomeres-Structure, Function, and Regulation.</a> Exp Cell Res. 319 (2), 133-141 (2013).</li><li>MacNeil, D. E., Bensoussan, H. J., Autexier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomerase+Regulation+from+Beginning+to+the+End.">Telomerase Regulation from Beginning to the End.</a> Genes (Basel). 7 (9), 64 (2016).</li><li>Fouquerel, E., Opresko, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergence+of+The+Nobel+Fields+of+Telomere+Biology+and+DNA+Repair.">Convergence of The Nobel Fields of Telomere Biology and DNA Repair.</a> Photochem Photobiol. , (2016).</li><li>Bernadotte, A., Mikhelson, V. M., Spivak, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markers+of+Cellular+Senescence.+Telomere+Shortening+as+a+Marker+of+Cellular+Senescence.">Markers of Cellular Senescence. Telomere Shortening as a Marker of Cellular Senescence.</a> Aging (Albany NY). 8 (1), 3-11 (2016).</li><li>Opresko, P. L., 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-Associated+Aging+Disorders.">Telomere-Associated Aging Disorders.</a> Ageing Res Rev. , 1568-1637 (2016).</li><li>Chiodi, I., Mondello, C. <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+Telomerase+Stability+in+Human+Diseases+and+Cancer.">Telomere and Telomerase Stability in Human Diseases and Cancer.</a> Front Biosci (Landmark Ed). 1 (21), 203-224 (2016).</li><li>Blackburn, E. H., Epel, E. S., Lin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Telomere+Biology:+A+Contributory+and+Interactive+Factor+in+Aging,+Disease+Risks,+and+Protection.">Human Telomere Biology: A Contributory and Interactive Factor in Aging, Disease Risks, and Protection.</a> Science. 350 (6265), 1193-1198 (2015).</li><li>Lindqvist, D., 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=Psychiatric+Disorders+and+Leukocyte+Telomere+Length:+Underlying+Mechanisms+Linking+Mental+Illness+with+Cellular+Aging.">Psychiatric Disorders and Leukocyte Telomere Length: Underlying Mechanisms Linking Mental Illness with Cellular Aging.</a> Neurosci Biobehav Rev. 55, 333-364 (2015).</li><li>Bodelon, C., Savage, S. A., Gadalla, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeres+in+Molecular+Epidemiology+Studies.">Telomeres in Molecular Epidemiology Studies.</a> Prog Mol Biol Transl Sci. 125 (113), (2014).</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> Nat Protoc. 5 (9), 1596-1607 (2010).</li><li>Haussmann, M. F., Mauck, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TECHNICAL+ADVANCES:+New+strategies+for+telomere-based+age+estimation.">TECHNICAL ADVANCES: New strategies for telomere-based age estimation.</a> Mol Ecol Resour. 8 (2), 264-274 (2008).</li><li>Nussey, D. H., 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=Measuring+telomere+length+and+telomere+dynamics+in+evolutionary+biology+and+ecology.">Measuring telomere length and telomere dynamics in evolutionary biology and ecology.</a> Methods Ecol Evol. 5 (4), 299-310 (2014).</li><li>Delany, M. E., Krupkin, A. B., Miller, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+telomere+sequences+in+birds:+evidence+for+arrays+of+extreme+length+and+for+in+vivo+shortening.">Organization of telomere sequences in birds: evidence for arrays of extreme length and for in vivo shortening.</a> Cytogenet Cell Genet. 90 (1-2), 139-145 (2000).</li><li>Baerlocher, G. M., Vulto, I., de Jong, G., Lansdorp, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+and+FISH+to+measure+the+average+length+of+telomeres+(flow+FISH).">Flow cytometry and FISH to measure the average length of telomeres (flow FISH).</a> Nat Protoc. 1 (5), 2365-2376 (2006).</li><li>Sanders, J. L., Newman, A. B. <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+in+epidemiology:+a+biomarker+of+aging,+age-related+disease,+both,+or+neither.">Telomere length in epidemiology: a biomarker of aging, age-related disease, both, or neither.</a> Epidemiol Rev. 35, 112-131 (2013).</li><li>Parikh, D., Fouquerel, E., Murphy, C. T., Wang, 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=Telomeres+are+partly+shielded+from+ultraviolet-induced+damage+and+proficient+for+nucleotide+excision+repair+of+photoproducts.">Telomeres are partly shielded from ultraviolet-induced damage and proficient for nucleotide excision repair of photoproducts.</a> Nat Commun. 6, 8214 (2015).</li></ol>

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.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Biologics</td>
			<td style="width:116px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Rsa1, restriction enzyme</td>
			<td style="width:116px;">New England Biolabs, Inc</td>
			<td style="width:119px;">R0167S</td>
			<td style="width:167px;">10,000 U/mL, DNA digestion.&#160; Comes with Cutsmart Buffer (digestion buffer)</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Hinf1, restriction enzyme</td>
			<td style="width:116px;">New England Biolabs, Inc</td>
			<td style="width:119px;">R0155S</td>
			<td style="width:167px;">10,000 U/mL, DNA digestion.&#160; Comes with Cutsmart Buffer (digestion buffer)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Seakem GTG Agarose</td>
			<td style="width:116px;">Lonza</td>
			<td style="width:119px;">50071</td>
			<td style="width:167px;">electrophoresis grade agarose</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Optikinase&#160;</td>
			<td style="width:116px;">affymetrix</td>
			<td style="width:119px;">78334X 500 UN</td>
			<td style="width:167px;">T4 Polynucleotide Kinase</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SYBR Green Nucleic Acid Stain I</td>
			<td style="width:116px;">ThermoFisher Scientific</td>
			<td style="width:119px;">S7567</td>
			<td style="width:167px;">Syber Green</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">exactGene DNA Ladder 24- kB</td>
			<td style="width:116px;">Fisher Scientific</td>
			<td style="width:119px;">BP2580100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">C-Strand Probe (3&#39;-(G3AT2)4-&#39;5)</td>
			<td style="width:116px;">Integrated DNA Technologies</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Custom ordered DNA oligonucleotide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gamma-ATP-P32</td>
			<td style="width:116px;">Perkin Elmer</td>
			<td style="width:119px;">BLU502Z</td>
			<td style="width:167px;">Radioisotope</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Qiagen Blood and Cell Culture DNA Mini Kit</td>
			<td style="width:116px;">Qiagen</td>
			<td align="right" style="width:119px;">13323</td>
			<td style="width:167px;">DNA extraction kit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">GE Illustra Microspin G-25 column</td>
			<td style="width:116px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">27-5325-01</td>
			<td style="width:167px;">For purification of readioactive oligo probe</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ficoll 400</td>
			<td style="width:116px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">non-ionic synthetic polymer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Equipment/Software</td>
			<td style="width:116px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Owl A5 Gel electrophoresis system (20 cm x 25 cm)</td>
			<td style="width:116px;">ThermoFisher Scientific</td>
			<td style="width:119px;">A5</td>
			<td style="width:167px;">Gel electrophoresis</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Horizon 11.4 Gel electrophoresis system (11 cm x 14 cm)</td>
			<td style="width:116px;">Corel Life Sciences</td>
			<td align="right" style="width:119px;">11068020</td>
			<td style="width:167px;">Gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nylon mesh, 50, 12&#34; x 12&#34;</td>
			<td style="width:116px;">Ted Pellam, Inc</td>
			<td style="width:119px;">41-12105</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">GE Storage Phosphor Screens</td>
			<td style="width:116px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">28-9564-75</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphor Screen Exposure Cassette</td>
			<td style="width:116px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">63-0035-44</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isotemp Hybridzation Incubator</td>
			<td style="width:116px;">Fisher Scientific</td>
			<td style="width:119px;">13-247-20Q</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cylindrical hybridization tubes</td>
			<td style="width:116px;">Fisher Scientific</td>
			<td style="width:119px;">13-247-300</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">The Belly Dancer orbital shaker</td>
			<td style="width:116px;">Sigma-aldrich</td>
			<td style="width:119px;">Z768499-1EA</td>
			<td style="width:167px;">Orbital shaker</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Typhoon 9400</td>
			<td style="width:116px;">ABI</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">For imaging of gels and phosphor screens</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GraphPad Prism 6</td>
			<td style="width:116px;">GraphPad</td>
			<td style="width:119px;">Version 6.05</td>
			<td style="width:167px;">Graphing Program</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microsoft Excel</td>
			<td style="width:116px;">Microsoft</td>
			<td style="width:119px;">Office 365</td>
			<td style="width:167px;">Spreadsheet program</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nanodrop</td>
			<td style="width:116px;">Thermo Scientific</td>
			<td style="width:119px;">Nanodrp 2000</td>
			<td style="width:167px;">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 ...

The overall goal of this procedure is to determine the length of telomeres in eukaryotic cells using a modified version of a telomere restriction fragment analysis procedure, that's described by Doctors Jerry W.Shay, Woodring Wright, and colleagues. By providing data on the telomere length, the procedure can answer questions on cell biology, behavioral medicine, epidemiology, and infectious diseases, on the effects of age, stress, drug treatments, and infections. The main advantage of this technique is that it provides a direct measurement in kilobases of the average telomere length in a variety of cells.The genomic DNA samples to be analyzed were extracted from cells using a commercial DNA extraction kit as described in the text protocol. Perform the digestion of the genomic DNA in a final volume of 20 to 40 microliters. Combine the following in each microcentrifuge tube:three micrograms of genomic DNA;10x DNA digest buffer;one microliter of Rsa1 restriction enzyme;one microliter of Hinf1 restriction enzyme;and sterile, deionized water to the final volume.Centrifuge briefly to ensure that all components are in the bottom of the tube. Incubate the digestions at 37 degrees Celsius, for at least 16 hours. Begin this procedure by preparing 0.6%agarose solution as described in the text protocol.Prepare the gel electrophoresis system for gel casting. Attach the end gates to the gel casting tray. Once the agarose gel solution has cooled to 50 degrees Celsius, pour it into the gel casting tray, place a cone into the gel, and allow the gel to harden for 45 minutes uncovered at room temperature.Remove the end gates from the casting tray, and remove the comb from the gel. Fill the gel electrophoresis system, with electrophoresis buffer to the fill line, ensuring that the gel is completely submerged in the buffer. Load the wells of the gel with the DNA samples, leaving a blank well between samples.Add 10 microliters of a DNA ladder into the wells on the opposite ends of the gel. Run the gel electrophoresis at 150 volts for 30 minutes, to quickly move the DNA into the gel. After 30 minutes, adjust the voltage to 57 volts, and run for 18 to 24 hours.After the gel has run for 18 to 24 hours, turn off the elecctrophoresis source and remove the gel casting tray with the gel from the elecctrophoresis buffer. Slice of the top right corner of the gel to serve as a reference for the orientation of the gel. Cut a piece of filter paper to a size slightly larger than the gel.Place the filter paper on top of the gel in a casting tray, and allow the paper to soak up the excess solution on the gel. Carefully remove the air bubbles between the filter paper and the gel, using a pipet as a roller to squeeze the bubbles out through the sides. 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 pipet as a roller. Dry the gel at 50 degrees Celsius for two hours.Once the gel has dried, remove the plastic wrap and transfer the gel and filter paper to a glass dish containing 250 milliliters of deionized water. Carefully remove the filter paper, and incubate the gel at room temperature for 15 minutes. Lay the gel on top of a 12 by 12 inch nylon mesh.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. Combine 100 milliliters of 5x SSC and 12 microliters of green fluorescent nucleic acid stain, and place into the hybridization tube.Incubate for 30 minutes at 42 degrees Celsius in a hybridization oven with rotation. Protect the solution from light. After 30 minutes, remove the nylon mesh and gel from the hybridization tube.Roll open and place flat into a glass dish containing 250 milliliters of deionized water. Gently remove the nylon mesh. Image the gel using a phosphorimager with the indicated settings.Prepare the radioactive telomere probe following the facility's regulations for handling radioactivity. In the microcentrifuge tube combine the following:two microliters of telomere probe;2.5 microliters of 10x polynucleotide kinase reaction buffer;three microliters to ATP labeled on the gamma phosphate group with P32;four microliters of T4 polynucleotide kinase;and DNase free, sterile, deionized water to a final volume of 20 microliters. Briefly centrifuge the tube.Incubate the tube in a water bath at 37 degrees Celsius for one hour. Centrifuge the tube briefly, and incubate at 65 degrees Celsius for 20 minutes to stop the reaction. Load the radioactive probe solution into a prepared G25 chromatography column, and centrifuge for two minutes at 700 times g.Save the filtrate, which contains the radioactive telomere probe. Place the gel into a glass dish containing 250 milliliters of Denaturing Solution and incubate for 15 minutes at room temperature. Remove the Denaturing Solution and replace with 250 milliliters of sterile, deionized water.Incubate for 10 minutes on an orbital shaker at room temperature. Remove the deionized water. Replace with 250 milliliters of neutralization solution and incubate for 15 minutes at room temperature.Roll the gel into a nylon mesh, and place into the hybridization tube. Add 20 milliliters of the hybridization solution, and incubate in a hybridization oven at 42 degrees Celsius for 10 minutes with rotation. After 10 minutes, remove the hybridization solution and replace with 20 milliliters of fresh hybridization solution.Add the entirety of the telomere probe, and incubate in a hybridization oven at 42 degrees Celsius with rotation overnight. When the hybridization with the telomere probe is complete remove the gel and mesh from the hybridization tube, and place into a glass dish, containing 250 milliliters of 2x SSC. Unroll and remove the nylon mesh from the dish.Place the glass dish onto an orbital shaker and incubate for 15 minutes at room temperature. Remove the 2x SSC, replace with 250 milliliters of 0.1x SSC, plus 0.1%SDS solution, and incubate for 15 minutes on the orbital shaker at room temperature. Remove the 0.1x SSC plus 0.1%SDS solution.Replace with 250 milliliters of 2x SSC and incubate for another 15 minutes on the orbital shaker. Remove the gel from the 2x SSC, and wrap the gel in plastic wrap. Remove all bubbles and air pockets between the gel and the plastic wrap.Place the gel into an exposure cassette with a phosphor screen, and expose the phosphor screen to the gel overnight. On the following day, use a phosphor imager to image the exposed phosphor screen. The phosphor images are subsequently used for telomere length quantification as described in the text protocol.Three concentrations of DNA isolated from an immortalized human foreskin fibroblast cell line and human peripheral blood mononuclear cells were analyzed for telomere length. This phosphor image of the dried agerose gel stained with green fluorescent nucleic acid stain shows the location of the DNA ladder bands in lanes one and eight. The distance measured from the top of the gel to each molecular weight standard is indicated by blue arrows.Following hybridization with a telomere probe, the telomere bands appear as smears in each lane. Grids of 150 boxes, were calculated for each lane plus one background lane. The selected areas of analysis for each set of samples are shown.Separate background lanes marked B were selected for each set of samples, to ensure the background intensities were similar for each sample set. Average telomere length calculations revealed a higher average telomere length in the immortalized cell line than in human peripheral blood mononuclear cells. These results also demonstrate that the amount of genomic DNA analyzed in each lane is critical to obtaining a detectable signal following hybridization.Once mastered, this technique can be performed in 12 hours spread out over 2 1/2 days, with two overnight incubations if it's performed properly. While attempting this procedure, it's important to remember to use at least three micrograms of DNA for each sample, and to measure each sample in duplicate. After its development, this technique paved the way for researchers in the field of aging to explore mechanisms responsible for maintaining telomere length in different animal species, including humans.After watching this video, you should have a good understanding of how to directly determine the average length of telomeres themselves. Don't forget that working with radioactivity can be extremely hazardous, and precautions such as wearing lab coats and gloves should always be taken while performing this procedure.

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Research

JoVE Journal

Cancer Research

12.7K visualizações.  University of Pittsburgh. The overall goal of this procedure is to determine the length of telomeres in eukaryotic cells using a modified version of a telomere restriction fragment analysis procedure, that's described by Doctors Jerry W.Shay, Woodring Wright, and colleagues. By providing data on the telomere length, the procedure can answer questions on cell biology, behavioral medicine, epidemiology, and infectious diseases, on the effects of age, stress, drug treatments, and infections. The main advantage of this te...