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.

动物实验批准了在本·古里安大学（IL-39-08-2010）动物实验伦理委员会。 1，鼠脑蛋白提取<ol><li>制备各含在冰上5毫升林格氏溶液和地方管15×3管中。 </li><li>用异氟醚麻醉牺牲鼠标（注意 - 使用化学罩）10-20秒后颈椎脱位和即时斩首。 </li><li>从头骨取出脑和漂洗数秒用冰冷的林格氏溶液，以防止损坏的脑组织。 </li><li>使用手术刀片，从额皮层分离的小脑（CR）和脑干（BS）。接着，分离脑干，小脑和切断来自额叶皮层的额叶（FL）;除去来自额叶嗅球。将3个脑区中的指定15ml试管。 </li><li>均质化中的每个管中的组织。添加FRESH冰冷的林格氏液使每管含有相同的体积和离心800×g的7分钟。 </li><li>除去上清液，加入100μl的CHAPS裂解液，每管。拌匀，通过1毫升注射器用21G针头使溶液多次（〜10），并在冰上孵育30分钟。 </li><li>以13,000×g的各管的内容物转移到一个新的微量离心管中，离心分离30分钟。将上清转移到一个新的微型离心管中，并确定使用先前建立的方法的蛋白浓度。存储所述蛋白质提取物中的10微升等分试样在-80℃下。 </li></ol>仪器仪表，反应液和蛋白质计算稀释2 TRAP法准备<ol><li>制备标记的微离心管中的蛋白质提取物的稀释和端粒酶的反应缓冲液的适当的数。执行所有的移液使用过滤嘴，以防止污染的协议。 </li&gt; <li>解冻的冰以下解决方案:陷阱反应混合物10倍，TS引物，dNTP的年代，超纯水和蛋白提取物（见表格材料，试剂和试剂浓度）。 </li><li>稀释的蛋白提取物的溶液来使用超纯水（UPW）1微克/微升的最终浓度。 </li></ol> 3，端粒酶反应<ol><li>准备端粒酶反应混合物中加入以下为微型离心管:2微升陷阱组合（10X），1微升的TS引物（0.1微克），1微升的dNTP（10毫米）和15μL超纯水。对于多个样品，通过样品中，加1的数乘以上述卷。 </li><li>加入19微升反应混合物中的每个反应管中，加入1微升稀释蛋白提取物（1微克蛋白）向每管中以20微升的最终体积。 </li><li>将反应管在30℃下预热的水浴中放置45分钟。 </li></ol> 4，PCR反应<ol><li>十五英里n中的端粒酶反应结束前，解冻了以下解决方案:ACX底漆，钛Taq缓冲液，超纯水。 </li><li> 2.5微升钛Taq缓冲液（10X），1微升ACX底漆（0.1微克），2μL超纯水（1微升时，IC的使用）和0.5微升钛Taq酶:通过添加以下到微量离心管中准备PCR反应混合物（50X）。对于多个样品，通过样品加2的数量乘以上述卷。 </li><li>加入6微升的PCR混合到每个PCR管中，添加了端粒酶反应（20微升），每管的整个体积为26微升的最终体积。 </li><li>将PCR管到的PCR热循环仪，并运行以下程序: - 90℃ - 2分钟。 - 94°C - 30秒* - 60°C - 30秒* - 72°C - 45秒​​ - 72℃ - 2分钟。 商店的PCR产物于-20℃。 * = 34次</li></ol> 5，琼脂糖迷你GEL的制备，样品运行和凝胶染色<ol><li>加25毫升冷冻电泳缓冲液（TBE）和搅拌棒的烧杯中是这样的缓冲溶液的2-4倍的体积和在1.125克的高分辨率（HR）琼脂糖粉末缓慢洒而将溶液快速搅拌。取出搅拌棒，盖上保鲜膜，皮尔斯在一个小孔用于通风的烧杯中。热火在"低"电3分钟微波的烧杯中，然后切换到"高级"电力，热力的短脉冲，注意不要造成溢出的解决方案。请注意，当通过加热产生的泡沫变大，气泡和消失，几秒钟后，将琼脂糖已准备就绪。 </li><li>投凝胶到水平微型凝胶装置中，凝胶固化后在室温下使凝胶在4℃下在使用前放松20分钟。 </li><li>加载每个车道25微升样品陷阱（20微升的TRAP PCR产物5μL6X凝胶样染料）和运行在110 V 3小时，在4℃冷室。 </li><li>加入15微升10000核酸染色，原液至50ml双蒸水（DDW）用0.1 M氯化钠（0.29克）准备核酸染色3倍的工作方案。染色凝胶20分钟，轻轻摇动。 </li><li>使用紫外透射与302 nm的波长，以查看陷阱阶梯DNA条带。 </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.

在通过TRAP法对小鼠脑细胞端粒酶活性的分析包括4个主要步骤:1）蛋白质从脑组织2）特定地区开采端粒酶TS引物延伸 - 端粒酶反应3）的端粒酶反应产物PCR扩增4 ）的分离使用高分辨率琼脂糖凝胶对PCR产物。 这种改良TRAP法解决了上升，从传统的检测两大困难:使用放射性的dCTP的灵敏检测PCR产物和PAGE分离的DNA梯状的。高分辨率琼脂糖凝胶简化了端粒酶的反应产物的检测（将DNA梯）...

端粒酶是一种端粒酶的组成核糖核蛋白逆转录酶（TERT），端粒酶催化亚基和RNA组分（TERC）。端粒酶的规范作用是通过将重复序列（TTAGGG）的端粒末端，因此促进基因组的完整性，以及细胞的增殖1保持端粒的适当长度。其他角色归因于TERT在细胞中， 即它赋予对诱导细胞凋亡的各种损伤剂2，保护免受氧化应激3人间充质干细胞，并通过其关联到TIA1阳性RNA起着完全分化的神经元的促存活作用颗粒4。 TERT表达和活性，主要在体分裂的细胞，并在大多数类型的癌症，而酶的表达和活性水平在非分裂细胞5,6紧密调节。有研究显示，在第rodent脑，端粒酶活性检测不到就由出生后10天，而TERT mRNA的维持在较低水平到成年7。其他研究表明成年子脑室区，嗅球，海马内端粒酶活性，并且在成人小脑和皮层8。我们最近证明了增加在大脑和脊髓中端粒酶的表达和活性的新化合物作用于N-甲基-D-天冬氨酸（NMDA）的神经保护作用 - 注射的小鼠，延迟的肌萎缩性侧索硬化病的SOD1转基因的发作和进展小鼠和增加运动神经元的存活在这些小鼠9的脊髓。要充分了解端粒酶在神经元和大脑中的促存活作用，有必要开发一种简单和相对敏感的快速测定端粒酶活性在脑的各个区域的检测。 端粒ŘEPEAT扩增法（TRAP）是一个众所周知的敏感测定法结合端粒酶的典型活性和聚合酶链式反应（PCR）。在该试验中，端粒酶添加TTAGGG重复到端粒酶衬底（TS引物）的寡核苷酸，然后通过PCR扩增，产生6个碱基对（bp）的使用TS的反向引物的DNA梯- ACX 32 p标记的dCTP的被用于检测的PCR产物的量低。的DNA梯状条带是由测序聚丙烯酰胺凝胶电泳（PAGE）解决。两个DNA带的强度和DNA片段化的长度反映活性酶分子的数量和它们的持续合成能力分别为10。 这种传统的检测容纳了一些重大的困难:暴露于放射性物质，大和难以处理的测序PAGE和凝胶运行的时间消耗和放射性产物的膜曝光（过夜在这两种情况下）的危险。 </p&gt; 该协议提供了一种改进的无放射性的琼脂糖微凝胶基础的检测。该DNA 6 bp梯度是使用4.5％的高分辨率琼脂糖凝胶上分辨并使用该检测，实现高灵敏度的核酸染色。使用高分辨率的琼脂糖微凝胶和敏感核酸染色的组合提供了易于处理法，可以消除暴露于放射性的危险和显著缩短为3天总测定时间至几小时。

<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

端粒酶，核糖核蛋白，是负责维持端粒的长度，从而推动基因组的完整性，细胞增殖和寿命。此外，端粒酶免受氧化应激线粒体和赋予抗细胞凋亡，表明如神经元非有丝分裂的，高活性的细胞的尚存其可能的重要性。我们以前表明的新颖端粒酶的活化剂，以增加端粒酶活性和表达在各种小鼠的大脑区域，并保护运动神经元细胞免受氧化应激的能力。这些结果加强了端粒酶是参与神经元的各种病变的保护的概念。强调端粒酶在脑的作用，我们在这里比较端粒酶在雄性和雌性小鼠大脑的活性和其对年龄依赖性。 TRAP法是在各种组织或细胞系中检测端粒酶活性的标准方法。在这里，我们展示了analy端粒酶活性在小鼠大脑的三个区域的sis通过非变性使用CHAPS蛋白提取裂解缓冲后跟标准TRAP法的变形例。 在这两步法，内源性端粒酶拉长特定端粒酶衬底（TS引物）加入TTAGGG 6 bp的重复序列（端粒酶反应）。端粒酶反应产物进行PCR反应创造了6个基点为单位的DNA阶梯放大。的DNA梯状条带的分析是由4.5％的高分辨率琼脂糖凝胶电泳，然后用高灵敏度的核酸染色剂染色制成。 相比于使用32 p标记的放射性的dCTP的用于DNA检测和聚丙烯酰胺凝胶电泳对解决该DNA梯的传统TRAP法，该协议提供了一种无毒的时间节省TRAP法评价端粒酶活性在小鼠脑，展示出的能力检测端粒酶的差异E活动中的各种女性，雄性小鼠的大脑区域。

全细胞蛋白质提取物来自额叶（佛罗里达州），脑干（BS）和小脑（CR）的制备，来自于3的CD-1雌性和3的CD-1雄性小鼠以1和3个月大的年龄为改性TRAP法和3的CD-1雄性小鼠以2个月大的放射性TRAP法的年龄。端粒酶活性在1微克的蛋白质提取物检测采用改良TRAP法和2微克的蛋白质中提取的放射性TRAP法。由放射性TRAP法和由改性TRAP法检测端粒酶活性表明，端粒酶的DNA产物的更好的可视化和分辨率被观察到的改?...

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.

在小鼠大脑的各个区域端粒酶活性:非放射性端粒酶重复序列扩增法（TRAP）分析

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

telomerase-activity-various-regions-mouse-brain-non-radioactive

Research

JoVE Journal

Neuroscience

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

14.6K 次观看.  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.

视频: 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.

人端粒酶逆转录蛋白 rna 依赖性 rna 聚合酶活性的半定量检测

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

科研

遗传学

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脑组织中的高灵敏度检测5 - hydroxymethylcytosine

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

神经科学

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.

RNA原位杂交法定量分析小鼠脑切片中的替代前 mRNA 拼接方法

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.

从活细胞中的单端粒表达的癌症细胞克隆的生成, 以可视化含有端粒重复的 rna terra

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.

液滴数字 TRAP (ddTRAP): 对端粒重复放大协议的适应性, 以实现液滴数字聚合酶链反应

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

癌症研究

使用非放射性化学发光检测定量端粒长度

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.

作者聚焦：TAGGG 端粒长度测定性能参数的优化  

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端粒长度测定性能参数优化

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.

动物的基因分型快速其次是建立大脑神经元原代培养

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.

在组织变性/再生和癌症的研究利用小鼠诱导端粒酶等位基因

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

在小鼠大脑的各个区域端粒酶活性:非放射性端粒酶重复序列扩增法（TRAP）分析

摘要

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此视频中的章节

人端粒酶逆转录蛋白 rna 依赖性 rna 聚合酶活性的半定量检测

BALB / C脑组织中的高灵敏度检测5 - hydroxymethylcytosine

RNA原位杂交法定量分析小鼠脑切片中的替代前 mRNA 拼接方法

从活细胞中的单端粒表达的癌症细胞克隆的生成, 以可视化含有端粒重复的 rna terra

液滴数字 TRAP (ddTRAP): 对端粒重复放大协议的适应性, 以实现液滴数字聚合酶链反应

TAGGG端粒长度测定性能参数优化

TAGGG端粒长度测定性能参数优化

TAGGG端粒长度测定性能参数优化

动物的基因分型快速其次是建立大脑神经元原代培养

在组织变性/再生和癌症的研究利用小鼠诱导端粒酶等位基因