Name: modified terminal restriction fragment analysis for quantifying telomere length using in-gel hybridization
Uploaded: 2017-07-10T10:30:00.000+00:00
Duration: 11 min 29 s
Description: Watch this Scientific Journal Video about Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization at JoVE.com

저자는 부분적으로 P30CA047904 상을 수상한 Pittsburgh Cancer Institute Biobehavioral 종양학 공유 시설의 지원을 인정합니다.

<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>

저자는 공개 할 것이 없습니다.

전기 영동 후 게놈 DNA 번짐은 800 bp보다 높습니다. 이는 제한 효소가 결함이거나 추가 효소 (위에서 설명한 바와 같음)가 필요한 경우 발생할 수 있습니다. 두 번째 가능한 설명은 단백질이 여전히 DNA에 붙어 있다는 것입니다. 분광 광도계로 DNA 농도를 측정 할 때 260/280 비율은 약 1.8이어야합니다. 이 비율이 &lt;1.5 인 경우 제한 효소 소화를 방해 할 수있는 단백질 오염이 있습니다. DNA 샘플을 재조직...

텔로미어 (telomeres)는 염색체의 말단에 위치하고 있으며, 세포 유전 정보를 보호하는 데 중요한 역할을하는 TTAGGG (인간 염색체상의 서열)의 뉴클레오타이드 반복 서열이다 3 , 4 , 5 . 텔로미어는 수천 개의 염기쌍으로 DNA 복제 중에 나머지 염색체의 무결성을 보호하는 역할을합니다. 염색체 복제가 완벽하지 않아서 염색체의 끝이 완전히 복사되지 않습니다. 이러한 복제의 비효율은 &quot;최종 복제 문제&quot;로 불리고 각 복제 라운드 동안 일부 텔로미어 반복을 잃게됩니다 6 , 7 . 텔로미어 길이는 잃어버린 텔로미어 서열 8 , 9 를 대체하는 효소 인 텔로 머라 아제에 의한 세포 분열 이후에 회복 될 수있다. 그러나, 텔로 머라 아제는결과적으로, 시간이 지남에 따라 텔로미어는 짧아지고 결국 세포 노화가 일어난다. 텔로미어 단축으로 인해 텔로미어는 노화와 노화 관련 질병의 위험성을 나타내는 바이오 마커로 간주됩니다 11,12 . 또한 텔로미어 길이는 유전 적, 환경 적, 생활 양식의 요인 및 산화 스트레스와 같은 다른 자극에 영향을 받아 텔로미어 길이가 독성학, 역학 및 행동 연구에서 바이오 마커 역할을 할 수 있음을 나타냅니다 13 , 14 , 15 . 이 기사는 Mender and Shay 2 와 Kimura et al.이 채택한 modified terminal restriction fragment (TRF) 분석을 사용하여 텔로미어 길이를 측정하는 기술을 설명합니다 . 16 , Herbert, Shay 및 Wrig와 비슷하다.ht 1 과 Haussmann과 Mauck 17 . 전통적으로, TRF 분석은 서던 블롯 절차를 포함합니다. 서던 블롯은 텔로미어 길이를 연구하기위한 &quot;금 표준&quot;으로 간주되며 역학 연구에서 백혈구 텔로미어 길이를 검사하는데 적극적으로 사용됩니다 18 . 이 TRF 분석은 텔로미어 반복을 남겨두고 소화 된 게놈 DNA를 사용합니다. 그런 다음 DNA 단편을 겔 전기 영동으로 분리하고 변성시키고 니트로 셀룰로오스 막으로 옮긴다. 텔로미어 서열은 텔로미어 올리고 뉴클레오티드 프로브에 하이브리드 화된다. 분리 된 텔로미어를 멤브레인으로 옮기는 대신, 다른 TRF 기술은 DNA 변성의 유무에 관계없이 in-gel hybridization 기술을 사용합니다. 변성의 포함은 일부 종 19 에서보고 된 간질 텔로미어의 검출을 고려할 때 중요합니다. 변성 단계가없는 절차는 th단일 가닥 DNA는 말단 텔로미어에서 오버행하고 간질 텔로미어 서열 18 에는 결합하지 않을 것이다. TRF 분석 외에도 텔로미어 길이를 측정하는 몇 가지 다른 기술이 있습니다. 각각의 장점과 단점이 있습니다. Flow-FISH 기술은 유동 세포 계측법을 사용하여 별개의 세포 유형의 개별 세포의 평균 텔로미어 길이를 측정 할 수 있습니다 16 , 20 . Flow-FISH는 자동화를 통해 텔로미어에 고도로 특화된 프로브를 정확하게 표기하고 사람의 실수를 줄이는 한편 비용이 많이 들고 효율적이지 않으며 신선한 샘플과 숙련 된 기술자가 필요하기 때문에 전염병 연구에 대한 능력이 제한됩니다 16 . 또한,이 방법은 세포 집단의 염색체 수의 잠재적 변화를 설명하지 못한다. 또 다른 기술인 qPCR은 전염병 연구를위한 텔로미어 길이를 측정하는 수단으로 널리 받아 들여지고있다 <sup class = &quot;xref&quot;&gt; 16은 다른 방법에 비해 적은 양의 DNA에 대한 높은 처리량, 저비용 및 요구 조건 때문에 발생합니다. 그러나, qPCR은 단일 복사 유전자에 비례하여 평균 텔로미어 DNA 함량만을 측정 할 수 있으며, 따라서 평균 텔로미어 길이의 간접적 인 추정치이다. 또한 남부 연구와 비교하여 비교 연구에서 높은 변동 계수를 산출하여 의심스러운 정확도 21로 이어진다. 재단 절차에는 일부 연구에 대한 사용을 제한하는 자체 결함이 있습니다. TRF 기술은 다른 방법에 비해 많은 양의 DNA가 필요합니다 (시료 당 2 ~ 3 μg). 이것은 충분한 게놈 DNA가 수집 될 수 있고 분해 된 DNA 샘플에는 사용할 수없는 임상 샘플의 텔로미어 길이를 검사하는 기술의 적용을 제한합니다. 또한, TRF 분석은 값 비싸고 노동 집약적이어서 결과를 산출하는 데 4-5 일이 걸립니다. 그러나 TRF는 낮은 변동 계수를 산출합니다.재현성을 부여합니다. 이 프로토콜은 기존의 Southern blot ( in situ gel hybridization을 사용)과 다르지만, 두 기술은 근본적으로 같습니다. 여기에 제시된 기술은 Southern blot과 in-gel hybridization의 조합입니다. Southern blots의 DNA 변성 단계를 in-gel hybridization과 관련 짓습니다. 이 조합은 서던 블롯에 비해 향상된 프로브 신호 강도의 추가 이점을 가지며 우리 연구실 내에서 지속적으로 정량화 가능한 결과를 산출했습니다. 또한 화학 발광 탐침보다 방사능 탐침을 사용하면 신호 강도가 높아지며 phosphorimager로 시각화 및 정량화가 가능하여 사용자 친화적 인 TRF 분석이 가능합니다.

<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

텔로미어 길이를 측정하는 몇 가지 다른 기술이 있습니다. 각각의 장점과 단점이 있습니다. 전통적인 접근 방법 인 Telomere Restriction Fragment (TRF) 분석법은 게놈 DNA 샘플을 제한 효소로 소화시켜 텔로미어 DNA 반복 및 일부 서브 텔로미어 DNA를 남기는 DNA 하이브리드 화 기술을 사용합니다. 이들은 아가로 오스 겔 전기 영동에 의해 분리되고, 필터 멤브레인으로 옮겨지고, 화학 발광 또는 방사능으로 표지 된 올리고 뉴클레오티드 프로브에 하이브리드 화되어 텔로미어 제한 단편을 시각화한다. 이 접근법은 PCR과 같은 다른 기술보다 많은 양의 DNA를 필요로하지만 세포 집단의 텔로미어 길이 분포를 측정 할 수 있으며 절대 킬로베이스로 표현 된 측정을 허용합니다. 이 원고는 텔로미어 길이를 결정하기위한 수정 된 DNA 하이브리드 화 과정을 보여줍니다. 게놈 DNA는 먼저 제한 효소 (절단하지 않는 게놈텔로미어), 아가 로스 겔 전기 영동으로 분리 하였다. 이어서, 겔을 건조시키고 DNA를 변성시키고 방사성 표지 된 올리고 뉴클레오티드 프로브 에 인 시츄 하이브리드 화시킨다. 이 in situ 하이브리드 화는 텔로미어 DNA의 손실을 피하고 신호 강도를 향상시킵니다. 하이브 리다이 제이션 후에, 겔은 형광체 스크린을 이용하여 영상화되고, 텔로미어 길이는 그래프 프로그램을 사용하여 정량화된다. 이 절차는 Drs의 실험실에서 개발되었습니다. Woodring Wright와 Jerry Shay는 텍사스 대학교 남서부 대학교 1 학년 2 학년입니다. 여기에서는 몇 가지 수정 사항을 포함하여이 절차에 대해 자세히 설명합니다.

세포주 BJ-hTERT (telomerase immortalized human foreskin fibroblasts)와 사람 말초 혈액 단핵 세포 (PBMCs)로부터 분리 된 3 가지 농도의 DNA (1, 2 및 3μg)를 텔로미어 길이에 대해 분석 하였다. 표 1 은 각 DNA 샘플의 레인 할당을 보여줍니다. 그림 1 은 젤의 핵산 형광 얼룩을 보여줍니다. 분자량 표준이 명확하게 나타납니다. 겔 상단에서 각 분자량 표준까지?...

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

이 기사에서는 텔로미어 길이의 신속하고 효율적인 직접 측정을 제공하는 수정 된 터미널 제한 단편 분석을 사용하여 텔로미어 길이를 정량화하기위한 자세한 프로토콜을 논의합니다. 이 기술은 텔로미어 길이를 정량화하기 위해 DNA의 다양한 세포 공급원에 적용될 수 있습니다.

In-gel Hybridization을 이용한 Telomere 길이 정량을위한 수정 된 terminal restriction fragment 분석

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 조회수.  University of Pittsburgh.  이 기사에서는 텔로미어 길이의 신속하고 효율적인 직접 측정을 제공하는 수정 된 터미널 제한 단편 분석을 사용하여 텔로미어 길이를 정량화하기위한 자세한 프로토콜을 논의합니다. 이 기술은 텔로미어 길이를 정량화하기 위해 DNA의 다양한 세포 공급원에 적용될 수 있습니다. 

비디오: 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.

Telomere G - 오버행 구조의 시험 Trypanosoma brucei</em

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

연구

면역학 및 감염병학

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.

수정 된 Blumgart Pancreaticojejunostomy와 복 강경 Pancreatoduodenectomy

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

암 연구학

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.

터치 인쇄물 세포학을 사용 하 여 병 적인 임상 표본에서 높은-품질 종양 DNA를 간단 하 고 빠른 방법

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.

Telomeric 반복 포함 된 RNA 테라에서 살아있는 세포에서 단일 Telomere 표현 시각화 하 암 세포 클론의 세대

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

유전학

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.

물방울 디지털 트랩 (ddTRAP): Telomere 반복 증폭 프로토콜의 적응은 디지털 중 합 효소 연쇄 반응에 액 적

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

<meta charset="utf8"></head><body>인간 집단 연구에서 정확한 텔로미어 길이 평가

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.

<meta charset="utf8"></head><body>저자 스포트라이트: 트라우마가 세포 노화에 미치는 영향 탐구

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.

Monochrome Multiplex Quantitative PCR 텔로미어 길이 측정 

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

Non-Radioactive Chemiluminescence Detection을 사용한 텔로미어 길이 정량화

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.

저자 스포트라이트: TAGGG 텔로미어 길이 분석의 성능 파라미터 최적화  

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.

TAGGG 텔로미어 길이 분석의 성능 매개변수 최적화

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...

In-gel Hybridization을 이용한 Telomere 길이 정량을위한 수정 된 terminal restriction fragment 분석

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