这项工作是由新加坡NRF奖学金（NRF-2011NRF-NRFF001-025）以及CBRG（NMRC / CBRG / 0070/2014）的资助。

注:在本研究的上下文中感兴趣的RNA为AR 3&#39;UTR的片段。 1.生物素标记的RNA的制备<ol><li>以获得的RNA，使用商业试剂盒和以下生产商的说明11通过PCR扩增T7-AR-低聚，随后通过体外转录。 注:引物用于PCR列于表4:T7-AR-F和T7-AR-R。 </li><li>对于非特异性结合控制，通过PCR扩增的萤火虫萤光素酶（FL）的DNA psiCHECK-2的质粒，随后通过体外转录的部分序列。 注:引物用于PCR列于表4:hluc（+） - T7探针-F和hluc（+） -探针- R 表4模板和引物进行PCR扩增的序列。 </li><li>对于50微升的PCR反应，混合50纳克DNA（寡核苷酸或质粒），4微升的dNTP（每个人的dNTP为2.5毫米），5微升10X反应缓冲液，1微升2.5 U /＆＃181;微升DNA聚合酶，2微升10μM的正向引物，2微升10μM反向引物，和无核酸酶水。 <ol><li>使用下列程序运行在热循环仪进行PCR扩增。第1步:95℃2分钟一个循环;第2步:35个循环的95℃30秒，55℃30秒和72℃30秒（AR）或60秒（佛罗里达州）; STEP3:10分钟，72℃的一个周期。 </li></ol></li><li>合成使用以下制造商的说明11 的体外转录试剂盒的生物素化的RNA。在该实验中，使用PCR产物作为用于RNA合成的模板。对于一个20微升的体外转录反应，混合200纳克的PCR扩增的DNA，2微升每个单独的NTP的，1微升10mM的生物素的CTP，2微升10×反应缓冲液，和2μlT7 RNA聚合酶。孵育在37℃下该混合物4小时。 </li><li>在37＃以下体外转录，用1微升2单位/微升DNA酶治疗反应混合物176℃持续15分钟，以降解模板DNA。最后，稀释生物素化的RNA，以60微升为随后的纯化的总体积。 </li><li>纯化生物素化的RNA，通过使用以下制造商的说明12纯化柱以除去盐和未掺入的核苷酸。测量RNA浓度，并储存于-80℃用于后续的下拉测定。 </li><li>至在90℃下确保在RNA-蛋白质相互作用，热加入50μl生物素化的RNA（50时）感兴趣的RNA适当的二级结构2分钟，冰浴2分钟，然后用50μl2×RNA结构缓冲液（ 表提供3），并允许将RNA在RT折叠30分钟13,14。 </li></ol> 2. RNA结合的珠的制备注:使用磁立场的磁珠和上清液分离。 <ol><li>通过简短vorte悬浮在原始小瓶链菌素包被的磁珠兴下列制造商的说明。转移150微升重新悬浮珠子到1.5ml管中。放置在磁体的管1分钟。弃上清。保持珠沉淀。 注:在RNA Pull-down实验，由原来的小瓶等分悬浮颗粒时，使用的检测之后印迹50微升珠和200微升珠化验后MS。 </li><li>通过重新悬浮珠粒料用1ml制造商推荐的1×W＆的B（结合和洗涤）缓冲液（ 表3）洗一次珠，随后在旋转器在室温下旋转管5分钟。弃上清。保持珠沉淀。 </li><li>通过重新悬浮珠粒料用400μl缓冲液A缓冲液A灭活珠上的RNase，洗珠两次，每次是含有0.1M NaOH和0.05 M氯化钠（ 表3）的碱性溶液。旋转管，在室温下在旋转器2分钟。弃上清。保持珠沉淀。 </li> &lt;li&gt;要通过重新悬浮珠粒料用400μl缓冲液B（ 表3），接着在一旋转器在室温下旋转管2分钟从珠除去氢氧化钠，洗珠两次，每次的时间。弃上清。保持珠沉淀。 </li><li>用300微升2倍W＆B缓冲液重悬珠颗粒，分割珠平等，转移成三个1.5毫升管（每管100微升珠）。供应含珠管100微升核糖核酸酶自由水，100微升生物素化FL RNA和100​​微升生物素化AR 3&#39;UTR RNA，分别为。 </li><li>孵育各胎圈混合物在室温下用旋转1小时。放置管上的磁铁1分钟。丢弃所有上清。保持珠颗粒。 </li><li>通过重新悬浮用400μl1×W＆B缓冲液各胎圈粒料洗涤珠两次，每次，随后在一旋转器在室温下旋转管5分钟。 </li><li>放置管上的磁铁1分钟。丢弃所有上清。重悬每个珠子PELLE吨，50微升核隔离缓冲液（ 表3）。 </li></ol> 3.前脂肪细胞的分离和脂肪生成诱导<ol><li>正如在以前的出版物5描述，剖析从肩胛间棕色脂肪垫前脂肪细胞（C57BL6野生型小鼠，3周龄）。 </li><li>生长并在体外分化前脂肪细胞5。细胞准备用于分化开始后4-5天的下游应用。 </li></ol>从初级脂肪细胞的细胞裂解物的制备4 注:确保细胞裂解液的制备所有的程序都在冰制成，和所有缓冲区，在冰上冷却。冰上预寒意DOUNCE玻璃均质。 <ol><li>成长并在15 cm的培养皿5（参见3.2节）区分脂肪前体细胞。每道菜都提供了足够的细胞一个RNA pull-down实验。在第4天或分化，丢弃细胞培养基的第5天，和洗涤策LLS一旦与1X PBS。 </li><li>收获细胞，加入10-15毫升1×PBS中至细胞，刮去离心200 XG收集在50ml管中的细胞，并沉淀细胞在RT 5分钟。用1毫升吸管弃上清。 </li><li>在2毫升悬浮细胞沉淀低渗缓冲液（ 表3），并在冰上孵育细胞15分钟。转移细胞到15毫升DOUNCE玻璃匀浆和剪切细胞机械地在冰上用20笔。 </li><li>沉淀细胞核离心3300 xg离心15分钟，在4℃。保持冰为进一步利用细胞核沉淀（参见4.6节）。转移上清液至1.5毫升管中，添加3M氯化钾到上清液以20,000 xg离心以获得150mM的终浓度，并旋转，在4℃下15分钟。 </li><li>离心后，小心地用1毫升吸管除去lipidlayeron顶部，并收集上清液作为细胞质细胞裂解物。 </li><li>重悬细胞核沉淀（见4.4）在1毫升核隔离缓冲。 ŧ转让（BOT）的细胞核用1毫升吸管15毫升DOUNCE玻璃匀浆，并机械上用100笔划冰剪切核。沉淀通过离心核碎片以20,000 xg离心15分钟，在4℃，并收集上清液作为核细胞裂解物。 </li><li>无论是结合了核和细胞质的细胞裂解物，或单独使用每个分数，下游下拉应用程序。在此演示实验中，细胞裂解物的这两个级分合并以1:1的体积比为1:1。 </li><li> RNase抑制剂添加到该未分级的细胞裂解物，以获得0.5U /微升的最终浓度。抛开100微升细胞裂解液作为免疫印迹15输入控制。 </li></ol> 5.绑定和RBP洗脱<ol><li>分裂细胞裂解物分成三个等份（在1.5ml管1毫升细胞裂解物），并与空白孵育，佛罗里达的RNA共轭和AR 3&#39;UTR的RNA共轭珠粒（见2.8节），分别为3小时，在4℃ C轮换。 </li><li>放置管上的磁铁1分钟。使用1毫升吸管，随后RNA分离从上清液来确认RNA的完整性收集上清液。通过使用以下制造商的说明（ 表2）的酸性硫氰酸胍-苯酚溶液分离RNA。通过分析核糖体RNA的28S和18S亚基评估RNA的完整性。保持冰珠颗粒。 </li><li>通过重新悬浮每个珠子粒料用含有40单位RNA酶抑制剂和0.25％IGEPAL，随后在旋转器在室温下旋转管2分钟1毫升核隔离缓冲器洗涤磁珠六次，每次。对于使用的磁珠和上清液分离磁立场。丢弃所有上清。保持冰珠颗粒。 </li><li>使用以下步骤，从免疫印迹珠洗脱RNA-蛋白复合物。首先，在悬浮75微升核隔离缓冲每个珠子颗粒;然后，加入25微升4倍免疫印迹装载BUFFER（含有SDS），得到100微升的总体积。最后，在95℃下煮沸此100μl的溶液珠5分钟。 注意:使用以下步骤，从MS珠洗脱RNA-蛋白复合物。第1步:重悬在100μl洗脱缓冲液（ 表3）每个珠子粒料;第2步:孵育珠样品在室温下用旋转2-3小时; STEP3:离心机，并收集含蛋白质的上清液; STEP4:提交的MS之前集中蛋白质溶液20-30微升。 </li><li>离心机每100微升珠样品以12,000×g离心30秒，在4℃，并且收集上清液。分装15微升上清免疫印迹分析（见6.1和6.2）。探测用稀释的HuR小鼠单克隆抗体的得到的膜（1:1,000稀释度），过夜。 </li></ol> 6.免疫印迹的RBP验证<ol><li>独立的限制性商业惯例，通过SDS-PAGE使用标准技术。 </li><li>我们开展通过探测与感兴趣的RNA相关的限制性商业惯例用标准技术船尾印迹。 注:在这个演示实验中，使用了的HuR蛋白的免疫检测的HuR鼠单克隆抗体。 </li><li>孵育稀释的HuR抗体所得膜:在5％（1 1000稀释）w / v的脱脂奶粉，1×TBS， 0.1％ 吐温- 20日4 °下缓慢摇动过夜。用1×TBST洗膜三次。在室温下1小时的第二抗体（山羊抗 - 小鼠IgG-HRP）孵育膜。洗膜。制定并记录信号。 </li></ol>

<ol>
	<li>Huot, M. &. #. 2. 0. 1. ;., 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=The+Sam68+STAR+RNA-binding+protein+regulates+mTOR+alternative+splicing+during+adipogenesis.">The Sam68 STAR RNA-binding protein regulates mTOR alternative splicing during adipogenesis.</a> Mol Cell. 46 (2), 187-199 (2012).</li><li>Sun, L., 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=Long+noncoding+RNAs+regulate+adipogenesis.">Long noncoding RNAs regulate adipogenesis.</a> Proc Natl Acad Sci U S A. 110 (9), 3387-3392 (2013).</li><li>Engreitz, J. 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=The+Xist+lncRNA+exploits+three-dimensional+genome+architecture+to+spread+across+the+X-chromosome.">The Xist lncRNA exploits three-dimensional genome architecture to spread across the X-chromosome.</a> Science. 341 (6147), 1237973 (2013).</li><li>Zhao, X. Y., Li, S., Wang, G. X., Yu, Q., Lin, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+long+noncoding+RNA+transcriptional+regulatory+circuit+drives+thermogenic+adipocyte+differentiation.">A long noncoding RNA transcriptional regulatory circuit drives thermogenic adipocyte differentiation.</a> Mol Cell. 55, 372-382 (2014).</li><li>Alvarez-Dominguez, J. R., 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=De+novo+reconstruction+of+adipose+tissue+transcriptomes+reveals+long+non-coding+RNA+regulators+of+brown+adipocyte+development.">De novo reconstruction of adipose tissue transcriptomes reveals long non-coding RNA regulators of brown adipocyte development.</a> Cell Metab. 21, 764-776 (2015).</li><li>Adams, D. J., Beveridge, D. J., van der Weyden, L., Mangs, H., Leedman, P. J., Morris, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HADHB,+HuR,+and+CP1+bind+to+the+distal+3-untranslated+region+of+human+renin+mRNA+and+differentially+modulate+renin+expression.">HADHB, HuR, and CP1 bind to the distal 3-untranslated region of human renin mRNA and differentially modulate renin expression.</a> J Biol Chem. 278 (45), 44894-44903 (2003).</li><li>Yeap, B. B., 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+binding+of+HuR+and+poly(C)-binding+protein+to+a+conserved+UC-rich+motif+within+the+3&#180;+untranslated+region+of+the+androgen+receptor+messenger+RNA.">Novel binding of HuR and poly(C)-binding protein to a conserved UC-rich motif within the 3&#180; untranslated region of the androgen receptor messenger RNA.</a> J Biol Chem. 277, 27183-27192 (2002).</li><li>K&#246;nig, J., Zarnack, K., Luscombe, N. M., Ule, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-RNA+interactions:+new+genomic+technologies+and+perspectives.">Protein-RNA interactions: new genomic technologies and perspectives.</a> Nat Rev Genet. 13, 77-83 (2012).</li><li>Ferr&#232;, F., Colantoni, A., Helmer-Citterich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+protein-lncRNA+interaction.">Revealing protein-lncRNA interaction.</a> Briefings in Bioinformatics. , 1-11 (2015).</li><li>McHugh, C. A., Russell, P., Guttman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+comprehensive+experimental+identification+of+RNA-protein+interactions.">Methods for comprehensive experimental identification of RNA-protein interactions.</a> Genome Biol. 15 (1), 203 (2014).</li><li>Lee, Y. S., 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=AU-rich+element-binding+protein+negatively+regulates+CCAAT+enhancer+binding+protein+mRNA+stability+during+long+term+synaptic+plasticity+in+Aplysia.">AU-rich element-binding protein negatively regulates CCAAT enhancer binding protein mRNA stability during long term synaptic plasticity in Aplysia.</a> Proc Natl Acad Sci U S A. 109 (38), 15520-15525 (2012).</li><li>Hermann, M., Cermak, T., Voytas, D. F., Pelczar, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+genome+engineering+using+designer+nucleases.">Mouse genome engineering using designer nucleases.</a> J Vis Exp. (86), (2014).</li><li>Tsai, M. C., 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=Long+Noncoding+RNA+as+Modular+Scaffold+of+Histone+Modification+Complexes.">Long Noncoding RNA as Modular Scaffold of Histone Modification Complexes.</a> Science. 329 (5992), 689-693 (2010).</li><li>Wan, Y., 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=Landscape+and+variation+of+RNA+secondary+structure+across+the+human+transcriptome.">Landscape and variation of RNA secondary structure across the human transcriptome.</a> Nature. 505, 706-709 (2014).</li><li>Li, 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=Identification+of+mRNA+binding+proteins+that+regulate+the+stability+of+LDL+receptor+mRNA+through+AU+rich+elements.">Identification of mRNA binding proteins that regulate the stability of LDL receptor mRNA through AU rich elements.</a> J Lipid Res. 50 (5), 820-831 (2009).</li><li>McHugh, C. 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=The+Xist+lncRNA+interacts+directly+with+SHARP+to+silence+transcription+through+HDAC3.">The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3.</a> Nature. 521 (7551), 232-236 (2015).</li><li>Chu, C., 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=Systematic+Discovery+of+Xist+RNA+Binding+Proteins.">Systematic Discovery of Xist RNA Binding Proteins.</a> Cell. 161 (2), 404-416 (2015).</li><li>Chu, C., Spitale, R. C., Chang, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technologies+to+probe+functions+and+mechanisms+of+long+noncoding+RNAs.">Technologies to probe functions and mechanisms of long noncoding RNAs.</a> Nat Struct Mol Biol. 22 (1), 29-35 (2015).</li><li>Zhao, J., 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=Genome-wide+identification+of+Polycomb-associated+RNAs+by+RIP-seq.">Genome-wide identification of Polycomb-associated RNAs by RIP-seq.</a> Mol Cell. 40 (6), 939-953 (2010).</li></ol>

lncRNAs和限制性商业惯例在健康和疾病的重要角色，但这些分子的分子机制知之甚少。与lncRNA分子相互作用的蛋白的鉴定是朝着阐明的调节机制的关键步骤。通过将RNA下拉测定系统限制性商业惯例的富集是基于在溶液中相互作用的复杂，使得从细胞裂解物蛋白可使用结合于链霉抗磁珠的生物素化的RNA诱饵被选择性地提取的捕获。其他一些方法，如啁啾，图表，RAP可以达到同样的目的，但这些方法?...

A correction to the author list was made to: <a href="http://www.jove.com/video/54207/detection-rna-binding-proteins-vitro-rna-pull-down-adipocyte">Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture</a>.
The author list has been updated from:
Qianfan Bai1*, Zhiqiang Bai1*, Lei Sun1,2
1Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School
2Institute of Molecular and Cell Biology, Singapore
*These authors contributed equally
to:
Qianfan Bai1*, Zhiqiang Bai1*, Shaohai Xu3, Lei Sun1,2
1Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School
2Institute of Molecular and Cell Biology, Singapore
3Division of Bioengineering, School of Chemical &#38; Biomedical Engineering, Nanyang Technological University
*These authors contributed equally

A correction to the author list was made to: Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture.

Erratum: Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

限制性商业惯例是结合在细胞中的双链或单链RNA和参与形成RNA-蛋白复合物的蛋白质。限制性商业惯例可以结合多种RNA分子，包括mRNA和长非编码RNA（lncRNAs），并在发挥转录后水平的影响力。限制性商业惯例和lncRNAs在脂肪发展逐渐成为新的监管机构和功能1,2,3。理解RBP-的机制和细胞途径lncRNA介导的调节，它通常需要检测特定RNA分子和一个或多个限制性商业惯例之间的相互作用，并且，有时，以识别RNA的蛋白伴侣的全谱成绩单。然而，该实验可以是具有挑战性由于脂质的脂肪细胞的高含量。这里所描述的RNA的下拉协议可被用于检索来自原代培养4,5的脂肪细胞裂解物的特定RNA的蛋白质的合作伙伴。 理这个原山口总结如下。的RNA诱饵是在体外从DNA模板转录，生物素标记和缀合到链霉抗包被的磁珠4。所述RNA诱饵与细胞裂解物孵育以允许RNA-蛋白质复合物，其随后被拉下来的磁性支架的形成。更具体地，该RNA下拉协议如下所述使用的RNA片段从AR 3&#39;UTR作为诱饵检索的HuR蛋白质，普遍表达RBP势必的mRNA 6,7的3&#39;UTR，从theadipocytelysate原代培养。为了测试该测定的结合特异性，非相关RNAas以及空白链霉珠被包括作为对照。此协议是与蛋白质印迹或质谱（MS）兼容，以确认一个特定RBP的捕获或识别捕获的限制性商业惯例，分别为8的全剧目。 几种技术可用于研究RNA-蛋白质相互作用秒。揭示通过一个给定的RBP，RIP（RNA免疫沉淀）和CLIP（UV交联和免疫沉淀）结合的RNA可以应用。与此相反，以确定一个给定RNA的蛋白质伙伴，RNA下拉，啁啾（通过RNA纯化染色质分离），图表（RNA靶捕获杂交分析）和RAP（RNA反义纯化）都可以应用。在与后来者相比，RNA下拉技术花费较少的精力来设置。它可以用来捕获蛋白在体外 和体内 。 RNA的下拉，这在技术上是比其体外对应更具挑战性， 在体内的方法，通过在细胞中的交联保留RNA-蛋白质相互作用，捕获的从细胞中感兴趣的适体-标记的RNA，和随后检测结合的限制性商业惯例。 RNA的下拉可以用于充实低丰度限制性商业惯例，并且分离和鉴定在控制细胞调节9,10具有多样功能作用的RNA-蛋白质复合</s了&gt;。

<table border="0" cellpadding="0" cellspacing="0" width="857">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:499px;">Major materials used in this study.</td>
			<td style="width:177px;"></td>
			<td style="width:109px;"></td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEGAscript kit</td>
			<td>&#160;Ambion</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotin-14-CTP&#160;</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NucAway spin columns&#160;</td>
			<td>Ambion</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dynabeads M-280 Streptavidin&#160;</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HuR (3A2) mouse monoclonal antibody (sc-5261)&#160;</td>
			<td>Santa Cruz Biotechnology</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-mouse IgG-HRP(sc-2005)&#160;</td>
			<td>Santa Cruz Biotechnology</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hypure Molecular Biology Grade Water (nuclease-free)&#160;</td>
			<td>HyClone</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffer Saline (1&#215;PBS)</td>
			<td>&#160;HyClone</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase inhibitor&#160;</td>
			<td>Bioline</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease inhibitor&#160;</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pfu Turbo DNA polymerase&#160;</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRIsure</td>
			<td>&#160;Bioline</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Major equipment used in this study.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorvall Legend Micro 21R centrifuge&#160;</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorvall Legend X1R centrifuge&#160;</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bio-Rad T100 thermal cycler&#160;</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nanodrop 2000&#160;</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BOECO rotator Multi Bio RS-24&#160;</td>
			<td>BOECO,Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein gel electrophoresis and blotting apparatus&#160;</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DynaMag-2 magnet&#160;</td>
			<td>Life Technologies</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of rna-binding proteins by in vitro rna pull-down in adipocyte culture

RNA结合蛋白（限制性商业惯例）正在成为在脂肪的发育和功能的调节层。限制性商业惯例通过影响靶mRNA的稳定性和翻译效率发挥在转录后水平的基因表达的调控中起关键作用。 RNA的下拉技术已被广泛用于研究RNA-蛋白质相互作用，这是必要阐明该机制底层限制性商业惯例&#39;以及长的非编码RNA"（lncRNAs）函数。然而，高血脂丰度在脂肪细胞构成在进行该实验中一个技术挑战。这里详细的RNA下拉协议是初级脂肪细胞培养进行了优化。从雄激素受体的（AR）3&#39;非翻译区含作为一个例子来说明如何检索其RBP合作伙伴的HuR蛋白质，脂肪细胞从一个lystate富含腺苷酸尿苷酸-elementwas（3&#39;UTR）的RNA片段。可以应用这里描述的方法来检测之间的相互作用限制性商业惯例和非编码RNA，以及间限制性商业惯例和编码RNA。

在这个演示实验中，AR RNA片段作为诱饵捕捉到它的结合蛋白的HuR。两者FL的RNA诱饵即非相关的HuR蛋白，以及未缀合的链霉空白珠的等分试样用作阴性对照。 RNA-蛋白的相互作用，可能会发生无论是在细胞核或细胞质中，并且此下拉协议可以应用到无论是总或分级（核或细胞质）细胞裂解物。通过Western印迹的蛋白质的分析表明，15微升未分级细胞裂解物的样品（见4.8节），如...

Watch this Scientific Journal Video about Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture at JoVE.com

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

的RNA下拉协议这里优化用于检测RNA结合蛋白（限制性商业惯例）和非编码以及编码RNA之间的相互作用。从雄激素受体（AR）的RNA片段作为例子来演示如何从初级的棕色脂肪细胞的lystate检索其RBP。

RNA结合蛋白的检测通过<em&gt;体外</em在脂肪细胞培养&gt; RNA下拉

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

detection-rna-binding-proteins-vitro-rna-pull-down-adipocyte

Research

JoVE Journal

Biology

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

23.5K 次观看.  Duke-NUS Medical School. 的RNA下拉协议这里优化用于检测RNA结合蛋白（限制性商业惯例）和非编码以及编码RNA之间的相互作用。从雄激素受体（AR）的RNA片段作为例子来演示如何从初级的棕色脂肪细胞的lystate检索其RBP。 

视频: Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

科研

生物学

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

激活细胞凋亡的细胞色素的细胞质显微注射 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Pull-down of Calmodulin-binding Proteins

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the calcium-binding protein known as calmodulin (CaM)1,2. CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca2+ (Ca2+-CaM) as opposed to its Ca2+-free state (ApoCaM)3.
While most target proteins bind Ca2+-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin4, neurogranin (Ng)5, and certain myosins6. These proteins have been shown to play important roles in presynaptic function7, postsynaptic function8, and muscle contraction9, respectively. Their ability to bind and release CaM in the absence or presence of Ca2+ is pivotal in their function. In contrast, many proteins only bind Ca2+-CaM and require this binding for their activation. Examples include myosin light chain kinase10, Ca2+/CaM-dependent kinases (CaMKs)11 and phosphatases (e.g. calcineurin)12, and spectrin kinase13, which have a variety of direct and downstream effects14.
The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca2+-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca2+-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function8,15. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM.
Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca2+, using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca2+ on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca2+/CaM signaling and proteins that interact with CaM.

Calmodulin (CaM) pull-down assay is an effective way to investigate the interaction of CaM with various proteins. This method uses CaM-sepharose beads for efficient and specific analysis of CaM-binding proteins. This provides an important tool to explore CaM signaling in cellular function.

钙调蛋白结合蛋白的下拉

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

在体外转录和Gaussia荧光素酶基因的上限由HeLa细胞转染

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA纯化的染色质隔离（CHIRP）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

荧光检测病毒RNA<em&gt;原位</em&gt;杂交（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster whole embryos, manually dissected larval/adult tissues or in vitro cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged.

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

增强Northern印迹检测小RNA种类的<em&gt;果蝇</em

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures (MAAC)

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments for T2D, more physiologically relevant in vitro adipocyte models are required. This study describes a new technique to isolate and culture mature human adipocytes. This method is entitled MAAC (membrane mature adipocyte aggregate culture), and compared to other adipocyte in vitro models, MAAC possesses an adipogenic gene signature that is the closest to freshly isolated mature adipocytes. Using MAAC, adipocytes can be cultured from lean and obese patients, different adipose depots, co-cultured with different cell types, and importantly, can be kept in culture for 2 weeks. Functional experiments can also be performed on MAAC including glucose uptake, lipogenesis, and lipolysis. Moreover, MAAC responds robustly to diverse pharmacological agonism and can be used to study adipocyte phenotypic changes, including the transdifferentiation of white adipocytes into brown-like fat cells.

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures MAAC

Membrane mature adipocyte aggregate culture (MAAC) is a new method to culture mature human adipocytes. Here we detail how to isolate adipocytes from human adipose and how to set up MAAC.

利用膜成熟腺细胞聚合培养（MAAC）的人类成熟腺细胞的分离与培养

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments ...