Name: telomerase activity in the various regions of mouse brain: non-radioactive telomerase repeat amplification protocol (trap) assay
Uploaded: 2014-09-02T13:00:00.000+00:00
Duration: 10 min 14 s
Description: Watch this Scientific Journal Video about Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay at JoVE.com

This work was partially supported by B.G. Negev technology and Linda Powers's contribution.

Hayvan deneyleri Ben-Gurion Üniversitesi&#39;nde (IL-39-08-2010) hayvan deneyleri için etik kurul tarafından onaylandı. 1. Fare Beyin Protein Ekstraksiyonu <ol><li> Her bir buz üzerinde 5 mi Ringer çözeltisi ve yeri içeren tüplerde 15 ml 3 tüpleri hazırlayın. </li><li> (DİKKAT - kimyasal kaput kullanmak) İsofloran anestezi kullanarak fare Kurban servikal dislokasyon ve hemen dekapitasyon 10-20 sn. </li><li> Kafatası beyin çıkarın ve beyin dokusunda hasar görmesini önlemek için buz gibi soğuk Ringer solüsyonu ile bir kaç saniye için durulayın. </li><li> Cerrahi bıçak kullanılarak, frontal korteks beyincik (CR) ve sapı (BS) ayırır. Sonra, beyin sapından beyincik ayırmak ve frontal korteks, frontal lobu (FL) kesilmiş; frontal lob koku ampulü çıkarın. Belirlenen, 15 ml tüpler içinde 3 beyin bölgelerinin yerleştirin. </li><li> Her bir tüp içinde doku homojenize edilir. Fr EkleESH, buz soğukluğundaki Ringer solüsyonu, her bir tüp, 800 xg da 7 dakika için aynı hacim ve santrifüj içerir. </li><li> Süpernatantı ve her tüp CHAPS lizis tamponu 100 ul ekleyin. , Bir 21 G iğne ile bir 1 ml şırınga ile çözelti, birkaç kez (~ 10) geçirilerek, iyice karıştırın ve 30 dakika boyunca buz üzerinde inkübe edilir. </li><li> 13,000 x g&#39;de 30 dakika boyunca yeni bir mikro santrifüj tüpüne ve santrifüj tüpünün her aktarın. Yeni bir mikro santrifüj tüpüne aktarılır ve süpernatan, önceden belirlenmiş bir yöntem kullanarak protein konsantrasyonunu belirleyin. -80 ° C&#39;de 10 ul alikolar içinde protein muhafaza edilir. </li></ol> Araçların, Reaksiyon Çözümleri ve Protein Sulandırma Hesaplamaları 2. TUZAK Testi Hazırlık <ol><li> Protein ekstresi seyreltilerde ve telomeraz reaksiyon tamponu için etiketli mikro-santrifüj tüplerine uygun sayıda hazırlayın. Kontaminasyonu önlemek için filtre ipuçlarını kullanarak protokoldeki tüm pipetlenmesini gerçekleştirin. </li&gt; <li> TRAP Reaksiyon karışımı 10x TS astar, dNTP&#39;ler, Ultra saf su ve protein ekstreleri (reaktif ve reaktif konsantrasyonları için malzeme tabloya bakınız): buz aşağıdaki çözümleri çözülme. </li><li> Ultra saf su (Ultra saf su) ile 1 mg / ml nihai konsantrasyona kadar bir protein ekstresi çözeltisi ile seyreltilir. </li></ol> 3. Reaksiyon Telomeraz <ol><li> Bir mikro santrifüj tüpüne aşağıdakiler eklenerek telomeraz reaksiyonu karışımı hazırlayın: 2 ul TRAP karışımı (10x), 1 ul TS primer (0.1 ug), 1 ul dNTPs (10 mM) ve 15 ul UPW. Çoklu örnekler için, numuneler, artı 1 sayısına göre yukarıda sözü edilen birimler çarpın. </li><li> Her reaksiyon tüpüne, reaksiyon karışımı 19 ul ve 20 ul son hacim için, her bir tüpe seyreltilmiş protein ekstresi (1 ug protein) 1 ul. </li><li> 45 dakika süre ile, 30 ° C&#39;de önceden ısıtılmış bir su banyosu içinde, reaksiyon tüpleri yerleştirin. </li></ol> 4. PCR Reaksiyonu: <ol><li> Onbeş milACX astar, Titanyum Taq Buffer, UPW: n telomeraz reaksiyon sonundan önce, aşağıdaki çözümleri eritin. </li><li> 2.5 ul Titanyum Taq Buffer (10x), 1 ul ACX astar (0.1 ug), 2 ul (IC kullanıldığında 1 ul) UPW ve 0.5 ul Titanyum Taq Polimeraz: mikro-santrifüj tüpüne aşağıdaki ekleyerek PCR reaksiyon karışımı hazırlayın (50x). Çoklu örnekler için, numuneler artı 2 sayısına göre yukarıda sözü edilen birimler çarpın. </li><li> PCR 6 ul her bir PCR tüpü 26 ul karıştırmak ve bir son hacim için her bir tüpe telomeraz reaksiyonu (20 ul), tüm birimi ekleyin. </li><li> Bir PCR termo döngü cihazı için PCR tüpleri yerleştirin ve aşağıdaki programı çalıştırın: - 90 ° C - 2 dk. - 94 ° C - 30 san. * - 60 ° C - 30 san. * - 72 ° C -. 45 sn * - 72 ° C - 2 dk. -20 ° C&#39;de muhafaza PCR ürünleri. * = 34 devir </li></ol> 5. Agaroz Mini-gel Hazırlama, Örnekleri Koşu ve Jel Boyama <ol><li> 25 ml soğuk tampon maddesi elektroforezi (TBE) ve tampon solüsyonu 2-4 kez hacminin yavaş yavaş yüksek çözünürlüklü (HR) agaroz toz halinde 1,125 g çözelti hızlı bir şekilde karıştırılır iken serpmek, bir cam kaba, bir karıştırma çubuğu ilave edin . Karıştırma çubuğu çıkarın, havalandırma için küçük bir delik plastik bir şal ve delgi ile beher kaplayın. Daha sonra 3 dakika boyunca &quot;düşük&quot; güç mikrodalga beher ısıtın ve &quot;Yüksek&quot; güç anahtarı ve dökülmesini neden değil itina ile kısa bakliyat çözüm ısıtın. Isıtılarak oluşturulan köpük, birkaç saniye sonra, büyük kabarcıklar haline gelir ve kaybolur, agaroz hazır olduğuna dikkat edin. </li><li> Jel, oda sıcaklığında katılaşan sonra jel, kullanımdan önce 4 ° C&#39;de 20 dakika boyunca soğuk izin yatay mini jel cihazına jel döküm. </li><li> 25 TUZAK numune ul (20 ul TRAP PCR ürünlerinin ve 5 ul 6x jel yükleme boyası) ile her şerit yerleştirin ve 11 çalıştırmak4 ° C soğuk oda içinde 3 saat boyunca 0 V. </li><li> 15 ul 10,000X nükleik asit leke, 0.1 M NaCl (0.29 g) ile 50 ml iki defa destillenmiş su (DDW) stok çözeltisi eklenerek çalışma çözeltisi nükleik asit lekesi 3x hazırlayın. Nazik çalkalama ile 20 dakika boyunca jel Leke. </li><li> TRAP merdiven DNA bantları görmek için 302 nm dalga boyunda UV transilluminator kullanın. </li></ol>

<ol>
	<li>Urquidi, V., Tarin, D., Goodison, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+telomerase+in+cell+senescence+and+oncogenesis.">Role of telomerase in cell senescence and oncogenesis.</a> Annu Rev Med. 51, 65-79 (2000).</li><li>Cong, Y., 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=Actions+of+human+telomerase+beyond+telomeres.">Actions of human telomerase beyond telomeres.</a> Cell Res. 18, 725-732 (2008).</li><li>Tichon, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+protection+by+novel+telomerase+activators+in+mesenchymal+stem+cells+derived+from+healthy+and+diseased+individuals.">Oxidative stress protection by novel telomerase activators in mesenchymal stem cells derived from healthy and diseased individuals.</a> Curr.Mol. Med. 13, 1010-1022 (2013).</li><li>Lannilli, F., Zalfa, F., Gartner, A., Bagni, C., Dotti, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+TERT+associates+to+RNA+granules+in+fully+mature+neurons:+Role+in+the+translational+control+of+the+cell+cycle+inhibitor+p15INK4B.">Cytoplasmic TERT associates to RNA granules in fully mature neurons: Role in the translational control of the cell cycle inhibitor p15INK4B.</a> PloS one. 8, e66602 (2013).</li><li>De Jesus, B. B., Blasco, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+of+telomerase+activation+in+extending+health+span+and+logevity.">Potential of telomerase activation in extending health span and logevity.</a> Curr opinion in Cell Biology. 24, 1-5 (2012).</li><li>Blackburn, E., Collins, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomerase:+An+RNP+enzyme+synthesizes+DNA.">Telomerase: An RNP enzyme synthesizes DNA.</a> Cold Spring Harb Perspect Biol. 3, 1-9 (2011).</li><li>Klapper, W., Shin, T., Mattson, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+telomerase+activity+and+TERT+expression+during+brain+development+in+mice.">Differential regulation of telomerase activity and TERT expression during brain development in mice.</a> J. Neurosci. Res. 64, 252-260 (2001).</li><li>Caporaso, G. L., Lim, D. A., Alvarez-Buylla, A., Chao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomerase+activity+in+the+subventricular+zone+of+adult+mice.">Telomerase activity in the subventricular zone of adult mice.</a> Mol. Cell Neurosci. 23, 693-702 (2003).</li><li>Eitan, E., 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=Novel+Telomerase-increasing+compound+in+mouse+brain+delays+the+onset+of+amyotrophic+lateral+sclerosis.">Novel Telomerase-increasing compound in mouse brain delays the onset of amyotrophic lateral sclerosis.</a> EMBO Mol. Med. 4, 313-329 (2012).</li><li>Kim, N. W., Wu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+quantification+and+characterization+of+telomerase+activity+by+the+telomeric+repat+amplification+protocol+(TRAP).">Advances in quantification and characterization of telomerase activity by the telomeric repat amplification protocol (TRAP).</a> Nuc. Acid Res. 25, 2595-2597 (1997).</li></ol>

The authors declare that they have no competing financial interests.

PCR 4 ile telomeraz reaksiyonu 3) telomeraz reaksiyon ürünlerinin amplifikasyonu - beyin dokusu 2) spesifik bölgelerinden 1) proteinlerinin ekstre Telomeraz suretiyle, primer uzaması TS: TRAP ile fare beyin telomeraz aktivitesi analizi 4 ana adımdan oluşur yüksek çözünürlüklü agaroz jel kullanılarak, PCR-ürünlerinin) ayrılması. DNA merdiveni ve PAGE PCR ürünleri ayırma hassas bir şekilde tespit radyoaktif dCTP &#39;lerin kullanımı: Bu değiştirilmiş TRAP yönteminin...

Telomeraz telomeraz bir ribonükleoprotein oluşan ters transkriptaz (TERT), telomeraz katalitik alt birimi, ve bir RNA bileşen (TERC) &#39;dir. Telomeraz kanonik rolü, bu nedenle genomik bütünlüğü ve hücre proliferasyonunu 1 teşvik telomerik uçlarına tekrar dizileri (TTAGGG) eklenerek telomerlerin uygun uzunluğunun korumaktır. Bu çeşitli zarar verici madde 2 ile uyarılan apoptoza karşı direnç kazandıran, yani başka roller de, hücrelerde TERT atfedilmiştir oksidatif stres 3 insan mezenkimal kök hücrelerini koruyan ve pozitif RNA TIA1 ile olan ilişkisi ile tam olarak farklılaşmış nöronların hayatta kalma yanlısı bir rol oynar granüller 4. Enzim ekspresyonu ve aktivitesi düzeyleri sıkı Bölünmeyen hücreler 5,6 olarak düzenlenmiş olsa da, TERT, ifade edilen ve daha çok somatik bölünen hücreleri ve kanser tiplerinin çoğunda aktif bir biçimde gösterilmiştir. Çalışmalar r göstermiştirTERT mRNA, erişkin 7 içine daha düşük düzeyde muhafaza edilirken Odent beyin, telomeraz aktivitesi, doğum sonrası 10. günde fark edilemez hale gelecektir. Diğer çalışmalar yetişkin alt-ventriküler bölge, koku ampul, hipokampus içinde telomeraz aktivitesini gösterdi ve yetişkin beyincik ve kortekste 8. , Enjekte edilen fareler SOD1 transjenik amiyotrofik lateral skleroz hastalığının başlamasını ve ilerlemesini yavaşlattığı - Yakın zamanda beyin ve omurilik telomeraz ekspresyonunu ve aktivitesini de artırır yeni bileşikler, N-metil-D-aspartat (NMDA) nöro-koruyucu etkiler üretmiştir göstermiştir fare ve bu farelerin 9 omurilikteki motor nöronların hayatta kalma artmıştır. Tam nöronlarda ve beyinde telomeraz pro survival rolünü anlamak için, beynin farklı bölgelerinde telomeraz aktivitesinin tespit edilmesi için basit, hızlı ve nispeten hassas bir analiz geliştirmek için gereklidir. Telomerik rEPEAT amplifıkasyon protokolü (TRAP) sıraları düzenlenmiştir telomeraz aktivitesi kanonik ve polimeraz zincir reaksiyonu (PCR) birleştiren bir iyi bilinen hassas bir deneydir. Bu deneyde, telomeraz TTAGGG telomeraz substrata TH ters primeri kullanılarak 6 baz çifti (bp) DNA merdiveni oluşturur PCR amplifikasyonu takiben (TH astar) oligonükleotid, tekrar ekler -. ACX 32P dCTP etiketli en tespit etmek için kullanılır PCR ürünlerinin, düşük miktarda. DNA merdiveni dizileme poliakrilamid jel elektroforezi (PAGE) ile çözümlenir. Her iki DNA bantların yoğunluğu ve DNA merdiven uzunluğu, aktif enzim moleküllerinin miktarını ve işlem kapasitesi, sırasıyla 10 yansıtmaktadır. Bu geleneksel tahlil bazı önemli zorluklar tutar: (gecede her iki durumda) sıralama PAGE ve jel çalışan zaman tüketimi ve radyoaktif ürünün filmi pozlama işlemek için büyük ve sert radyoaktif maddelere maruz kalma tehlikesi. </p&gt; Bu protokol, gelişmiş bir radyoaktif olmayan bir agaroz mini jel bazlı bir deney sağlar. DNA, 6 bp&#39;lik bir basamağı% 4.5 yüksek çözünürlüklü agaroz jel kullanılarak çözümlenir ve algılama son derece hassas bir nükleik asit leke kullanılarak başarılabilir. Agaroz mini yüksek çözünürlük jel ve duyarlı nükleik asit leke kullanılarak kombinasyonu, analizi kullanımı kolay sunar radyoaktiviteye maruz tehlikesini bertaraf eder ve önemli bir kaç saate kadar 3 gün genel deney süresini kısaltır.

<table><tbody><tr><td>TRAP Mix (10x)</td><td></td><td></td><td>Made manually, mix the following in UPW: 630 mM KCl, 200 mM Tris-HCl pH 8.2, 10 mM EDTA, 15 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 1 mg/ml BSA, 0.5% TWEEN (purchased from Sigma). Aliquots are frozen at -20 &amp;deg;C.</td></tr><tr><td>Ringer&#039;s solution</td><td></td><td></td><td>Made manually, mix the following in DDW: 124 mM NaCl, 3 mM KCl, 1.25 mM NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;H&lt;sub&gt;2&lt;/sub&gt;O, 2 mM MgSO&lt;sub&gt;4&lt;/sub&gt;, 26 mM NaHCO&lt;sub&gt;3&lt;/sub&gt;, 1.802 g/L D-(+)-glucose anhydrous.</td></tr><tr><td>Isoflurane</td><td>Minrad INC.</td><td>NDC 60307-110-10</td><td>Handle with care in a chemical hood</td></tr><tr><td>dNTPs (10 mM)</td><td>Sigma</td><td>D7295</td><td></td></tr><tr><td>TS primer (5&#039;-AATCCGTCGAGCAGAGTT-3&#039;)</td><td>Sigma</td><td></td><td>DNA oligos orderd in DRY format and UPW added according to requierd concentration</td></tr><tr><td>ACX primer (5&#039;-GCGCGGCTTACCCTTACCCTTACCCTAACC-3&#039;)</td><td>Sigma</td><td></td><td>DNA oligos orderd in DRY format and UPW added according to requierd concentration</td></tr><tr><td>Titanium Taq Polymerase/Buffer</td><td>Clontech</td><td>S1792/S1793</td><td></td></tr><tr><td>High Resolution agarose</td><td>Sigma</td><td>A4718</td><td>Any high quality high-resolution agarose may be sutible</td></tr><tr><td>Mini-gel apparatus</td><td>Bio-Rad</td><td>170-4466</td><td></td></tr><tr><td>Nucleic Acid Stain (GEL-RED)</td><td>Biotium</td><td>41003</td><td></td></tr><tr><td>IC primer (5&#039;-ATCGCTTCTCGGCCTTTT-3&#039;)</td><td>Sigma</td><td></td><td>DNA oligos orderd in DRY format and UPW added according to requierd concentration</td></tr><tr><td>CHAPS lysis buffer</td><td>Cell Signaling Technology</td><td>9852S</td><td>Add PMSF (protease inhibitor) to a final concentration of 1 mM</td></tr><tr><td>Ultra Pure Water (UPW)</td><td>Biological Industries</td><td>01-866-1B</td><td></td></tr><tr><td>CD-1 mice</td><td>HARLAN Laboratories INC.</td><td></td><td></td></tr></tbody></table>

telomerase activity in the various regions of mouse brain: non-radioactive telomerase repeat amplification protocol (trap) assay

Telomeraz, bir ribonükleoprotein, telomer uzunluğu muhafaza ve dolayısıyla genomik bütünlüğü, çoğalmasını ve ömrünü teşvik sorumludur. Nöronlar gibi mitotik olmayan, son derece aktif hücreler için hayatta kalan olası önemini göstermektedir, ilave olarak, telomeraz gibi oksidatif stresten mitokondri korur ve apoptoza direnç kazandırmaktadır. Daha önce çeşitli fare beyin bölgelerinde telomeraz aktivitesi ve ekspresyonunu artırmak ve oksidatif stres motor nöronlar hücreleri korumak için yeni telomeraz aktivatörlerinin yeteneğini gösterdi. Bu sonuçlar telomeraz çeşitli lezyonlar gelen nöronların korunması dahil olduğu görüşünü güçlendirmektedir. Beyinde telomeraz rolünü vurgulamak amacıyla, burada erkek ve dişi fare beyninde telomeraz aktivitesini ve yaş olan bağımlılığını karşılaştırın. TRAP yönteminin çeşitli dokular veya hücre hatlarında telomeraz aktivitesini tespit etmek için standart bir yöntemdir. Burada analı göstermekCHAPS kullanılarak protein çıkarımı ile denatüre edici olmayan fare beyninin üç bölgede telomeraz aktivitesi sis, standart TRAP modifikasyonu ile, ardından tampon lisis. Bu 2-aşamalı bir deneyde, endojen telomeraz TTAGGG 6 bp&#39;lik bir tekrar (telomeraz reaksiyonu) ekleyerek belirli bir alt-tabaka, telomeraz (TH primer) ilerlemektedir. Telomeraz reaksiyonu ürünleri 6 bp&#39;lik fazlalıklarının bir DNA merdiveni oluşturarak bir PCR tepkimesi ile yükseltilir. DNA merdiveni analizi son derece hassas bir nükleik asit boyası ile boyandı, ardından% 4.5 yüksek çözünürlüklü agaroz jel elektroforezi ile yapılır. 32P DNA tespiti ve DNA merdiveni çözülmesi için poliakrilamid jel elektroforez için radyoaktif dCTP etiketlenmiş kullanabilecektir geleneksel TRAP ile karşılaştırıldığında, bu protokol, fare beyninin telomeraz aktivitesini değerlendirmek yeteneğini göstermek için TRAP tasarruf toksik olmayan bir zaman sunar telomeras farklılıkları tespitÇeşitli dişi ve erkek fare beyin bölgesinde aktiviteye sahiptir.

Tüm hücre protein için 1 ve 3 aylık yaş 3, CD-1 erkek ve 3, CD-1 erkek fareler türetilen, frontal lob (FL), beyin sapı (BS), serebellum (CR) hazırlanmıştır modifiye TRAP tahlil ve radyoaktif TRAP eski 2 aylık yaşta 3 CD-1 erkek fareler. Telomeraz aktivitesi modifiye TRAP deneyi kullanılarak ayıkla 1 mcg proteinler tespit ve 2 mcg proteinler radyoaktif TRAP için ayıklamak edildi. Radyoaktif TRAP ile ve modifiye TRAP ile telomeraz aktivitesinin tespiti 1C genel DNA merdiveni <str...

Watch this Scientific Journal Video about Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay at JoVE.com

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

Fare Beyin Çeşitli Bölgelerinde Telomeraz Aktivite: Radyoaktif olmayan Telomeraz Amplifikasyon Protokolü tekrarlayın (TRAP) Deneyi

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

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Research

JoVE Journal

Neuroscience

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

14.6K Görüntüleme.  Ben-Gurion University of the Negev. Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

Video: Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase activity. Although TERT is named as a reverse transcriptase, structural and phylogenetic analyses of TERT demonstrate that TERT is a member of right-handed polymerases, and relates to viral RNA-dependent RNA polymerases (RdRPs) as well as viral reverse transcriptase. We firstly identified RdRP activity of human TERT that generates complementary RNA stand to a template non-coding RNA and contributes to RNA silencing in cancer cells. To analyze this non-canonical enzymatic activity, we developed RdRP assay with recombinant TERT in 2009, thereafter established in vitro RdRP assay for endogenous TERT. In this manuscript, we describe the latter method. Briefly, TERT immune complexes are isolated from cells, and incubated with template RNA and rNTPs including radioactive rNTP for RdRP reaction. To eliminate single-stranded RNA, reaction products are treated with RNase I, and the final products are analyzed with polyacrylamide gel electrophoresis. Radiolabeled RdRP products can be detected by autoradiography after overnight exposure.

Human telomerase reverse transcriptase (TERT) synthesizes not only telomeric DNA but also double-stranded RNA through RNA-dependent RNA polymerase activity. Here, we describe a newly established assay to detect RNA-dependent RNA polymerase activity of endogenous TERT.

İnsan revers transkriptaz protein RNA'ya bağımlı RNA polimeraz aktivitesinin yarı kantitatif algılama

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase ...

Genetik

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research. Mammalian DNA is enzymatically modified at the 5th carbon position of cytosine (C) residues to 5-mC, predominately in the context of CpG dinucleotides. 5-mC is amenable to enzymatic oxidation to 5-hmC by the Tet family of enzymes, which are believed to be involved in development and disease. Currently, the biological role of 5-hmC is not fully understood, but is generating a lot of interest due to its potential as a biomarker. This is due to several groundbreaking studies identifying 5-hydroxymethylcytosine in mouse embryonic stem (ES) and neuronal cells. Research techniques, including bisulfite sequencing methods, are unable to easily distinguish between 5-mC and 5-hmC . A few protocols exist that can measure global amounts of 5-hydroxymethylcytosine in the genome, including liquid chromatography coupled with mass spectrometry analysis or thin layer chromatography of single nucleosides digested from genomic DNA. Antibodies that target 5-hydroxymethylcytosine also exist, which can be used for dot blot analysis, immunofluorescence, or precipitation of hydroxymethylated DNA, but these antibodies do not have single base resolution.In addition, resolution depends on the size of the immunoprecipitated DNA and for microarray experiments, depends on probe design. Since it is unknown exactly where 5-hydroxymethylcytosine exists in the genome or its role in epigenetic regulation, new techniques are required that can identify locus specific hydroxymethylation. The EpiMark 5-hmC and 5-mC Analysis Kit provides a solution for distinguishing between these two modifications at specific loci. The EpiMark 5-hmC and 5-mC Analysis Kit is a simple and robust method for the identification and quantitation of 5-methylcytosine and 5-hydroxymethylcytosine within a specific DNA locus. This enzymatic approach utilizes the differential methylation sensitivity of the isoschizomers MspI and HpaII in a simple 3-step protocol. Genomic DNA of interest is treated with T4-BGT, adding a glucose moeity to 5-hydroxymethylcytosine. This reaction is sequence-independent, therefore all 5-hmC will be glucosylated; unmodified or 5-mC containing DNA will not be affected. This glucosylation is then followed by restriction endonuclease digestion. MspI and HpaII recognize the same sequence (CCGG) but are sensitive to different methylation states. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmC or 5-ghmC) at either cytosine blocks cleavage. MspI recognizes and cleaves 5-mC and 5-hmC, but not 5-ghmC. The third part of the protocol is interrogation of the locus by PCR. As little as 20 ng of input DNA can be used. Amplification of the experimental (glucosylated and digested) and control (mock glucosylated and digested) target DNA with primers flanking a CCGG site of interest (100-200 bp) is performed. If the CpG site contains 5-hydroxymethylcytosine, a band is detected after glucosylation and digestion, but not in the non-glucosylated control reaction. Real time PCR will give an approximation of how much hydroxymethylcytosine is in this particular site. In this experiment, we will analyze the 5-hydroxymethylcytosine amount in a mouse Babl/C brain sample by end point PCR.

The EpiMark 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a spe cific locus. The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing &beta;-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable site.

Balb / C Beyin Dokusu Yüksek Hassasiyet 5-hydroxymethylcytosine Algılama

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research.   Mammalian DNA is enzymatically ...

Nörobilim

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

Kantitatif analiz alternatif Pre-mRNA RNA In Situ hibridizasyon tahlil kullanarak fare beyin bölümlerde Uçbirleştirme

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

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.

Canlı hücreler içinde tek bir telomer üzerinden ifade Telomeric tekrar içeren RNA TERRA görselleştirmek için kanser hücre klonlar nesil

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

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.

Damlacık dijital tuzak (ddTRAP): telomere tekrar amplifikasyon protokolünün droplet dijital polimeraz zincir reaksiyonuna adaptasyon

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

Kanser Araştırmaları

Radyoaktif Olmayan Kemilüminesans Tespiti Kullanılarak Telomer Uzunluğunun Ölçülmesi

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.

Yazar Spotlight: TAGGG Telomer Uzunluk Testi'nin Performans Parametrelerinin Optimizasyonu  

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 Telomer Uzunluk Testi'nin Performans Parametrelerinin Optimizasyonu

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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Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

Hayvanlar Rapid Genotipleme Beyin Nöronlar İlköğretim Kültürler kurulması izledi

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

Utilizing Murine Inducible Telomerase Alleles in the Studies of Tissue Degeneration/Regeneration and Cancer

Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that cause tissue degeneration during ageing. Cancer, with incidence rates greatly increasing with age, is characterized by short telomere lengths and high telomerase activity. To study the roles of telomere dysfunction and telomerase reactivation in ageing and cancer, the protocol shows how to generate two murine inducible telomerase knock-in alleles 4-Hydroxytamoxifen (4-OHT)-inducible TERT-Estrogen Receptor (mTERT-ER) and Lox-Stopper-LoxTERT (LSL-mTERT). The protocol describes the procedures to induce telomere dysfunction and reactivate telomerase activity in mTERT-ER and LSL-mTERT mice in vivo. The representative data show that reactivation of telomerase activity can ameliorate the tissue degenerative phenotypes induced by telomere dysfunction. In order to determine the impact of telomerase reactivation on tumorigenesis, we generated prostate tumor model G4 PB-Cre4 PtenL/L p53L/L LSL-mTERTL/L and thymic T-cell lymphoma model G4 Atm-/- mTERTER/ER. The representative data show that telomerase reactivation in the backdrop of genomic instability induced by telomere dysfunction can greatly enhance tumorigenesis. The protocol also describes the procedures used to isolate neural stem cells (NSCs) from mTERT-ER and LSL-mTERT mice and reactivate telomerase activity in NSCs in vitro. The representative data show that reactivation of telomerase can enhance the self-renewal capability and neurogenesis in vitro. Finally, the protocol describes the procedures for performing telomere FISH (Fluorescence In Situ Hybridization) on both mouse FFPE (Formalin Fixed and Paraffin Embedded) brain tissues and metaphase chromosomes of cultured cells.

Telomere and telomerase play essential roles in ageing and tumorigenesis. The goal of this protocol is to show how to generate two murine inducible telomerase knock-in alleles and how to utilize them in the studies of tissue degeneration/regeneration and cancer.

Doku Dejenerasyonu / Yenilenme ve Kanser Araştırmaları Murin İndüklenebilir Telomeraz allelleri kullanılması

Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that ...

Fare Beyin Çeşitli Bölgelerinde Telomeraz Aktivite: Radyoaktif olmayan Telomeraz Amplifikasyon Protokolü tekrarlayın (TRAP) Deneyi

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İnsan revers transkriptaz protein RNA'ya bağımlı RNA polimeraz aktivitesinin yarı kantitatif algılama

Balb / C Beyin Dokusu Yüksek Hassasiyet 5-hydroxymethylcytosine Algılama

Kantitatif analiz alternatif Pre-mRNA RNA In Situ hibridizasyon tahlil kullanarak fare beyin bölümlerde Uçbirleştirme

Canlı hücreler içinde tek bir telomer üzerinden ifade Telomeric tekrar içeren RNA TERRA görselleştirmek için kanser hücre klonlar nesil

Damlacık dijital tuzak (ddTRAP): telomere tekrar amplifikasyon protokolünün droplet dijital polimeraz zincir reaksiyonuna adaptasyon

TAGGG Telomer Uzunluk Testi'nin Performans Parametrelerinin Optimizasyonu

TAGGG Telomer Uzunluk Testi'nin Performans Parametrelerinin Optimizasyonu

TAGGG Telomer Uzunluk Testi'nin Performans Parametrelerinin Optimizasyonu

Hayvanlar Rapid Genotipleme Beyin Nöronlar İlköğretim Kültürler kurulması izledi

Doku Dejenerasyonu / Yenilenme ve Kanser Araştırmaları Murin İndüklenebilir Telomeraz allelleri kullanılması