作者想感谢的技术援助，准备组织筐兰一，新泽西癌症研究所，博士。这项工作是由美国国立卫生赠款DK083425和DK070219研究院。

1。地高辛- 11 - UTP标记Riboprobe从一个PCR生成的模板合成<ol><li>为了合成一个特定基因riboprobe，使用Entrez基因（http://www.ncbi.nlm.nih.gov/sites/entrez）获得的基因的cDNA参考序列（的RefSeq） 。使用Primer3程序（http://frodo.wi.mit.edu/primer3/）5，设计基因特异性PCR引物对cDNA序列的3&#39; -地区。 PCR引物的选择推荐的参数描述（http://www.gudmap.org/Research/Protocols/Vezina/Riboprobe_Syn.html）。 </li><li>使用MegaBLAST计划（http://www.ncbi.nlm.nih.gov/genome/seq/BlastGen/BlastGen.cgi?taxid=10090）6，评估所选择的DNA序列的特异性。 DNA序列被认为是特定的时候，使用0.01期望阈值，它不配合RefSeq数据库中的其他序列。 </li><li> PCR扩增riboprobe模板。应该优化，以​​每个引物PCR反应的成分和热循环条件。一个典型的50μl反应体系包括：1X缓冲，2MM MgCl 2的 ，0.2MM dNTPs，1X Q的解决方案，1μg基因，2.5U的Taq DNA聚合酶，0.25μm的引物，无核酸酶的H 2 O一个典型的热循环协议包括变性94 ° C为2分钟，30秒94 ° C，40个周期，57 ° C为30秒，72 °，1分钟和10分钟在72℃最后延伸彗星。在PCR反应中使用的基因是从小鼠的泌尿生殖系统mRNA的合成。 </li><li>琼脂糖凝胶电泳分离PCR产物，纯化使用凝胶提取试剂盒，并量化分光纯化产物。预期收益率是1.2 - 3.6μg。 </li><li>转录成标记riboprobe的PCR产物。转录反应（40μL）包含：400ng纯化的PCR产物，1X核苷酸标签组合包含1X转录缓冲液，地高辛11双绞线，5U RNase抑制剂，80U T7 RNA聚合酶，无核酸酶的H 2 O 3 - 4小时在37℃，搅拌样品每次30min。 </li><li>使用Qiagen公司的RNeasy试剂​​盒，根据清理柱上的DNA酶消化RNA的指示，以净化riboprobes。用分光光度法定量riboprobes。预期收益率是4-20微克。评估探头质量1.5％的非变性琼脂糖凝胶电泳分离等分。高品质的探针迁移以最小的涂抹不同的波段。 </li><li>为了确保riboprobe明确承认其目标，包括第一原位杂交实验的阳性对照组织。 riboprobe的靶mRNA模式应该知道在这种积极的控制组织。 </li></ol> 2。振动切片机刀片的制备（基于先前所描述的协议）7 <ol><li>准备在4％的低熔点琼脂糖溶液磷酸缓冲液（PBS）。微波解决方案溶解琼脂糖和维护解决方案在62 ° C。 </li><li>准备从直径12mm Millicell小培养板取出膜以及插入和保留聚苯乙烯环使用琼脂糖模具聚苯乙烯环模。浸泡RNase抑制剂溶液过夜每次使用前的戒指。 </li><li>为了确保顺利的切割面，删除以下溶剂清洗，从威尔金森刀片表面的防锈剂和其他添加剂，在浓度为100％：石油醚，二甲苯，氯仿，甲醇，MilliQ水。双层刀片纵向分开成两个单叶片。 </li><li>坚持威尔金森刀片切片机刀片与乐泰胶。切片机刀片用于切割，它增加了刚性，威尔金森刀片。切片机刀片，应减少使用金属剪刀，一旦坚持威尔金森刀片长度，应抵消了3 - 4mm的威尔金森刀片的切削刃。 </li></ol> 3。夹层，存储和泌尿生殖系统组织切片的制备<ol><li>准备PBSTw解决方案（PBS含0.1％吐温™20和0.2mm的叠氮化钠，通过0.22μm的Stericup过滤®过滤装置）。解决方案可以提前做好准备，并储存在25 ° C </li><li>孵育新鲜解剖小鼠LUT（膀胱，骨盆尿道，以及相关的沃尔夫和Mϋllerian管派生结构）在4℃过夜°彗星在PBS中含4％多聚甲醛固定液。 </li><li>脱水洗涤10min后在25 °分级甲醇/ PBSTw（1:3，1:1，3:1 V / V）的解决方案系列中的彗星组织。在-20 ° C时，至少隔夜在100％甲醇的商店样品。存档的组织可能保持至少2yr。 </li><li>准备补水存档组织切片组织。 10min后洗净，在25 °一系列分级甲醇/ PBSTw C（3:1，1:1，1:3 V / V）的解决方案。 </li><li>解剖并丢弃约三分之二的膀胱，而使尿道三角地区最。 </li></ol> &lt;P类=“j​​ove_title”&gt; 4。泌尿生殖道组织中嵌入琼脂糖<ol><li>将聚苯乙烯环模，平坦的表面向下，25 ° C的普通玻璃显微镜玻片上。 </li><li>填写与62℃的琼脂糖溶液约2分钟冷却环模。 </li><li>从PBSTw删除LUT的组织和印迹吸水擦拭干。 </li><li>琼脂糖溶液转移到组织。 </li><li>东方的组织，以便它是中途暂停环模的顶部和底部之间，组织孵育4℃直至琼脂糖凝固的琼脂糖LUT的使用镊子。 </li><li>如果组织汇完全在琼脂糖凝固过程中，可以从琼脂糖和重新嵌入切除。在重新嵌入的过程中需要调整琼脂糖冷却时间。 </li></ol> 5。泌尿生殖系统组织切片随着振动切片机（基于先前所描述的协议） 7 <ol><li>安装在振动切片机的钢筋威尔金森刀片和叶片角度35 °。填写豪华标本用PBS和包装周围的标本浴湿冰浴。 </li><li>从环模中取出凝固的琼脂糖插件和杂交用吸水擦拭底部表面。确认组织的方向正确。组织的方向可以调整使用刀片斜角平边的琼脂糖插件。 </li><li>坚持琼脂糖插件安装如图所示的乐泰胶粘剂的磁盘到振动切片标本。 1A。 </li><li> vibratome插入试样安装磁盘。 </li><li>切片机切片厚度为50μm，速度为2，和刀片幅度调整到4，并开始切割组织切片。 </li><li>使用钝钳，每个组织切片（ 图1B）转移到24孔培养板以及包含冰冷0.5ML PBSTw。 </li><li>为了准备样品周围的每个组织切片（剩余的琼脂糖融化在原位杂交的过程中）琼脂糖的原位杂交，消费和删除所有关联的碎片。商店的部分达48小时，在4 °在PBSTw彗星。 </li></ol> 6。样品篮准备在原位杂交<ol><li>削减在100μL商标离心管底部。 </li><li>热管火焰切割边缘，直到塑料软化，然后用力按压到了0.5英寸的聚酯丝网广场中心的离心管。 </li><li>修剪多余的网格，使用到每个管盖了激烈的18号针头刺穿两个洞完成篮子准备（ 图1C） 。 </li><li>取出24孔板的盖子，对每口井为中心钻12毫米的孔。使用盖子转移样品筐之间的ISH的协议洗涤（ 图1D）。 </li></ol> 7。胚胎粉的制备原位杂交（基于先前所描述的协议） 8 <ol><li>正在评估组织部分相同的阶段，并储存在-80 ° C小鼠，收集小鼠胚胎组织放入陶瓷砂浆，淹没组织在液氮中冷冻组织，并使用杵研磨成细粉的组织。 </li><li>结合胚胎丙酮与4卷粉和同质化与几个招一个dounce匀浆。 </li><li>匀浆液转移到15ML的玻璃螺杆样品瓶，并提取一夜之间在4 ° C </li><li>球在5000RPM离心10min后胚胎粉在4 ° C。取出并丢弃上清液含脂。重悬新鲜丙酮4vol组织沉淀和提取2小时4 ° C。 </li><li>颗粒在5000RPM离心10min后的胚胎粉在4 ° C。取出上清液。 </li><li>空气干燥2号的Whatman滤纸上沉淀。粉碎颗粒细粉和储存在一个密封的玻璃小瓶，收益率在4 ° C。近似​​的收益率是50毫克每1克胚胎湿重的粉末。 </li></ol> 8。 原位杂交第1天<ol><li>预热预杂交液（50％甲酰胺，SSC的5倍，1％阻断试剂，酵母tRNA10μg/mL，10μg/mL肝素储存在-20 ° C）至60.5 ° C。该解决方案可提前做好准备，并储存在-20 ° C </li><li>准备一个小塑料存储容器灌装自来水约0.5加湿杂交腔。盖容器和预热至60.5 ° C。 </li><li>加入2ml PBSTw 24孔培养板内的水井。将样品在24孔板盖和转移组织切片，入筐（每筐多达10个路段已被用于）的孔筐。 </li><li>组织切片孵育30min后在25 ° C的6％H 2 O 2。这和所有后续孵化应进行轨道摇床轻轻摇动，除非另有说明。所有孵化一个ð洗24孔板进行，并使用了2mL/w​​ell的整体解决方案的体积。 </li><li>组织切片洗净，于25 °彗星在PBSTw 4 × 5分钟。 </li><li>孵育12分钟的组织切片在25 °在PBSTw彗星含有5μg/mL蛋白酶K </li><li>组织切片洗净，于25 °彗星在PBSTw 1 × 5分钟。 </li><li> 20分钟后修复组织切片在25℃的PBS彗星含有4％多聚甲醛和0.2％戊二醛。 </li><li>组织切片洗净2 25 × 5分钟°在PBSTw彗星。 </li><li>添加预热预杂交缓冲2mL/w​​ell和孵化组织切片内至少1小时的加湿杂交室为60.5 ° C。 </li><li>添加在每口井，并培育的组织切片，在加湿杂交室内过夜0.65μg标记riboprobe预杂交缓冲为60.5 ° C。 </li></ol> 9，2日在原位杂交<ol><li>准备杂交后的洗涤步骤如下解决方案：解决方案1（50％甲酰胺，SSC的5倍，1％SDS），解决方案2（10MM的Tris - HCl pH值7.5，0.5M氯化钠，0.1％吐温™20，0.2mm的叠氮化钠0.22μm的过滤），和解决方案3（2X SSC，50％甲酰胺）。这些解决方案可以提前做好准备。在-20 ° C和解决方案2的存储解决方案1和3是存储在25 ° C。贮存寿命的解决方案是至少3个月。 </li><li>组织切片洗净，为60.5 3 × 30分钟℃，预热解决方案1。在洗涤过程中使用湿盒。 </li><li>组织切片洗净60.5 1 × 10分钟° C预热的解决方案1/Solution 2（1:1 V / V）溶液。使用在洗湿盒。 </li><li>组织切片洗净2解决方案在25 ° C，4 × 10分钟。 </li><li>在37 °溶液C含有0.25μg/mL核糖核酸组织部分为15分钟。 </li><li>组织切片洗净，25 1 × 10分钟°与解决方案2℃（无核糖核酸）。 </li><li>组织切片洗净，25 1 × 10分钟° C的解决方案3，2 X1hr 60.5℃清洗解决方案3。使用在60.5℃洗湿盒。 </li><li> DIG标记riboprobes的免疫组化检测的准备下面的解决方案：组织封闭液（结核病，1X TBS的，10％羊血清，1％阻塞试剂，1％BSA，0.1％吐温™20，0.22μm的过滤），抗体稀释缓冲区（AD，1xTBS，5％羊血清，1％阻塞试剂，1％BSA，0.1％吐温™20，0.2mm的叠氮化钠，0.22™）抗体吸收液（AA，1xTBS，5％羊血清，1米过滤％阻断剂，1％BSA，6mg/mL胚胎粉），并TBSTw（1xTBS，0.1％吐温™20，0.2mm的叠氮化钠，0.22μm的过滤）。这些解决方案可以提前做好准备。 TBSTw是储存在25 ° C，所有其他的解决方案是储存在-20 ° C。 </li><li>洗3 × 10分钟在25 °与TBSTw彗星。 </li><li>孵育至少在2小时25 ° C的结核病缓冲区的组织切片。 </li><li>虽然组织结核病缓冲区的孵化，添加AA每200μL缓冲液1.1μL抗- DIG抗体，每孔。孵育机管局缓冲区+抗体至少在2小时4 ° C，然后在1分钟10000转离心。取出AA缓冲上清，将它添加到AD的缓冲液2ml。 </li><li>结核病缓冲区取出组织切片，并培育他们在4个湿盒过夜° C AD中含有抗体的缓冲区。 </li></ol> 10日在原位杂交。 <ol><li>准备彩色的开发解决方案NTMT（100MM的Tris - HCl pH值9.5，100mm的氯化钠，氯化镁2 50MM，0.2MM的叠氮化钠，0.22μm的过滤）。该解决方案可提前做好准备，并储存在25 ° C之前使用，添加2MM左旋咪唑和0.1％吐温™20。 </li><li>删除从井和存储解决方案，在4 ° C的抗体溶液（公元缓冲区+抗体）它可重复使用两个额外的时间。 </li><li>洗净组织8 × 10分钟25 °与TBSTw彗星含有2mm的左旋咪唑。 </li><li>小心地从篮子转移到培养皿中含有TBSTw组织。使用镊子去除可见的碎片。转移到干净的离心管组织。 </li><li>洗净组织1 × 10分钟在25 °与1ML NTMT。 </li><li>删除NTMT和一个含有50％NTMT（含2MM左旋咪唑）和50％BM紫，地方管，轻保护箱孵育25的混合物添加1mL/tube ° C。颜色的开发时间从数小时至数天不等。如果颜色是发展缓慢，100％的骨髓紫都可以使用。 </li><li> NTMT / BM紫色的解决方案显示器色彩的发展和变化，如果积累沉淀的晶体或如果它经历了从黄色到紫色的颜色变化。 （4-250小时），全彩色的发展后，洗净组织2 × 5分钟，25 °彗星含有2mm的左旋咪唑NTMT 1mL/tube。 </li><li>组织过夜孵育4 ° PBS 1mL/tube中的C含有4％的多聚甲醛后固定液。 </li><li>漂白的组织，在25 ° 30分钟的孵育组织PBSTw 1mL/tube中的C 含 3％H 2 O 2 </sUB&gt;。然后洗净组织25 1 × 10分钟° C在PBSTw 1mL/tube和储存在4℃PBS 1mL/tube中的C含4％多聚甲醛固定液后。 </li><li>组织切片装在载玻片上，coverslipped，和一个复式显微镜成像。 </li></ol> 11。代表性的成果： 琼脂糖LUT的组织的空间定位，决定组织切片的飞机。矢状部分，至少三分之二的膀胱切除，其余的LUT组织嵌入琼脂等，尿道中线琼脂糖插头（ 图1A）平坦的表面平行。在组织平面的一些小的调整可以由平边倒角的琼脂糖插件。从17.5dpc男性的LUT组织代表矢状切面，在这架飞机是面向如图1b所示。 样品篮，防止丢失和细腻的组织切片，在多天的ISH的过程中积累的灰尘和颗粒物。样品筐准备熔点聚酯丝网砍完一个1.5ml的离心管（ 图1C）。有一个小孔刺穿到每个样品篮盖，以方便流入和流出的花篮的解决方案流。样品篮暂停在ISH的解决方案，将它们放在12毫米孔钻成24孔板盖（ 图 1D ）。修改后的板盖，支持24孔板转移过程中的解决方案改变时，它们之间的篮子。 这是具有挑战性的限制在低丰度mRNA的检测所需时间的潜伏期长的非特异性背景染色。此外0.2mm厚叠氮化钠样本缓冲区，并随后将其过滤，通过0.22μm的过滤器出现限制背景染色（图 2 ）。使用这里介绍的方法，但不会出现背景染色明显的差别，样品时，在色彩的开发解决方案培养一个长期的时间段（ 图 3 ）。 <img src="/files/ftp_upload/2912/2912fig1.jpg" alt="图1"/> 图1。鼠标降低泌尿生殖道（LUT），组织切片和离心管一篮子为ISH的准备。一个LUT包含膀胱，骨盆尿道及相关沃尔夫和Mϋllerian管派生的结构）的一部分嵌入在4％低熔点琼脂糖的圆柱型插头。 （一）该插件是粘到样品安装盘和（二）用振动切片机切到50μm的部分。 （c）一个LUT的部分转移到微量离心管中篮，被刺穿了一个洞，入管的盖子和融合聚酯丝网管切底准备。 （四）的离心管插入12毫米孔钻成24孔板盖在缓冲溶液中悬浮在ISH的协议，使组织切片。箭头指示LUT的组织在琼脂糖插件。 <img src="/files/ftp_upload/2912/2912fig2.jpg" alt="图2"/> 图2团0.2mm厚叠氮化钠为0.22μm的过滤解决方案，提高组织的质量并降低背景染色。 17.5dpc雄性小鼠降低泌尿生殖道（LUTS）在矢状面厚度为50μm的切片。使用针对扭同源1探针原位杂交组织切片染色。 ISH的使用缓冲区是（A），0.22μm的过滤，并辅以0.2mm厚叠氮化钠（NaAz）或（B）与NaAz过滤并辅以。箭头指示的背景染色。在相同的放大倍率图像被抓获。结果是代表染色N = 3的垃圾的独立小鼠模式。 <img src="/files/ftp_upload/2912/2912fig3.jpg" alt="图3"/> 图3。背景染色强度不会出现长时间的颜色发展增加。 17.5dpc雄性小鼠降低泌尿生殖道（LUTS）在矢状面厚度为50μm的切片。第孵化chromagen染色液染色（一）9.5h使用探针承认的高丰度成绩单雌激素相关受体γ（Esrrg），（B）为43.5h使用探针承认的中等丰，通过原位杂交成绩单溴锌指结构域附近，2A（Baz2a），或（C）236H使用探针，认识到低丰度成绩单翅型MMTV整合位点的家庭，成员10A（Wnt10a）。在相同的放大倍率图像被抓获。结果是代表染色N = 3的垃圾的独立小鼠模式。

<ol>
	<li>Cunha, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+possible+influence+of+temporal+factors+in+androgenic+responsiveness+of+urogenital+tissue+recombinants+from+wild-type+and+androgen-insensitive+(Tfm)+mice.">The possible influence of temporal factors in androgenic responsiveness of urogenital tissue recombinants from wild-type and androgen-insensitive (Tfm) mice.</a> Journal of Experimental Zoology. 205, 181-181 (1978).</li><li>Cunha, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormonal,+cellular,+and+molecular+regulation+of+normal+and+neoplastic+prostatic+development.">Hormonal, cellular, and molecular regulation of normal and neoplastic prostatic development.</a> Journal of Steroid Biochemistry and Molecular Biology. 92, 221-221 (2004).</li><li>Price, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+aspects+of+development+and+structure+in+the+prostate.">Comparative aspects of development and structure in the prostate.</a> National Cancer Institute Monographs. 12, 1-1 (1963).</li><li>Cunha, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal-epithelial+interactions--I.+Induction+of+prostatic+phenotype+in+urothelium+of+testicular+feminized+(Tfm/y)+mice.">Stromal-epithelial interactions--I. Induction of prostatic phenotype in urothelium of testicular feminized (Tfm/y) mice.</a> Journal of Steroid Biochemistry. 14, 1317-1317 (1981).</li><li>Rozen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer3+on+the+WWW+for+general+users+and+for+biologist+programmers.">Primer3 on the WWW for general users and for biologist programmers.</a> Methods in Molecular Biology. 132, 365-365 (2000).</li><li>Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+greedy+algorithm+for+aligning+DNA+sequences.">A greedy algorithm for aligning DNA sequences.</a> Journal of Computational Biology. 7, 203-203 (2000).</li><li>Vezina, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dioxin+causes+ventral+prostate+agenesis+by+disrupting+dorsoventral+patterning+in+developing+mouse+prostate.">Dioxin causes ventral prostate agenesis by disrupting dorsoventral patterning in developing mouse prostate.</a> Toxicological Sciences. 106, 488-488 (2008).</li><li>Wilkinson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+messenger+RNA+by+in+situ+hybridization+to+tissue+sections+and+whole+mounts.">Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts.</a> Methods in Enzymology. 225, 361-361 (1993).</li></ol>

没有利益冲突的声明。

使用这里介绍的方法，它可以检测到在所有主要类型的细胞和胎儿的雄性和雌性小鼠的LUT，包括间质垫，尿路上皮，平滑肌，前列腺芽，射精管，及阴道组织的车厢的mRNA。在本议定书中所使用的50μm的部分有足够厚，以解决组织架构（如血管）的优势，但足够薄，以避免探头诱捕，这是一个方法论的问题，在整个安装ISH普遍遭遇。每一个新的riboprobe评估阳性对照组织，染色已在先前发表的研究报?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td>抗地高辛抗体Fab片段</td><td>罗氏应用科学部</td><td> 11214667001 </td></tr><tr><td>阻断试剂</td><td>罗氏应用科学部</td><td> 11096176001 </td></tr><tr><td> BM紫美联社衬底，沉淀</td><td>罗氏应用科学部</td><td> 11442074001 </td></tr><tr><td>牛血清白蛋白</td><td> Fisher Scientific则</td><td> BP1600 - 100 </td></tr><tr><td>细胞培养板，以及24 </td><td>康宁</td><td> 3524 </td></tr><tr><td>地高辛11 -双绞线</td><td>罗氏应用科学部</td><td> 1277073910 </td></tr><tr><td> dNTPs </td><td>罗氏应用科学部</td><td> 11969064001 </td></tr><tr><td>双刃刀片</td><td>威尔金森剑</td><td>经典模型</td></tr><tr><td> Eliminase核糖核酸卸妆</td><td> DECON实验室</td><td> 1102 </td></tr><tr><td>甲酰胺</td><td>西格玛</td><td> F5786 - 1L </td></tr><tr><td>凝胶提取试剂盒</td><td> Qiagen公司</td><td> 28704 </td></tr><tr><td>戊二醛，25％的H 2 O的解决方案</td><td>西格玛</td><td> G6257 - 100ML </td></tr><tr><td>肝素，钠盐</td><td>西格玛</td><td> H3393 </td></tr><tr><td>过氧化氢 ​​，30％的溶液中H 2 O </td><td> Fisher Scientific则</td><td> BP2633 - 500 </td></tr><tr><td>左旋咪唑</td><td>西格玛</td><td> L9756 </td></tr><tr><td>乐泰404快速设置瞬间粘合剂</td><td>汉高公司</td><td> 46551 </td></tr><tr><td>氯化镁</td><td> Fisher Scientific则</td><td> M33 - 500 </td></tr><tr><td>马来酸</td><td>西格玛</td><td> M0375 - 500G </td></tr><tr><td>离心管，1.5ml的</td><td> Biologix研究公司</td><td> BP337 - 100 </td></tr><tr><td> Millicell小文化板插入</td><td> Millipore公司</td><td> PICM01250 </td></tr><tr><td>分子研磨树脂</td><td> G - Biosciences公司</td><td> 786 - 138PR </td></tr><tr><td>多聚甲醛，4％的磷酸盐溶液缓冲液</td><td> Affymetrix公司</td><td> 19943 </td></tr><tr><td>磷酸盐缓冲液，无钙和镁</td><td> MP生物医学</td><td> ICN1760420 </td></tr><tr><td>聚酯网，33微米，12“× 24” </td><td>小零件公司</td><td> CMY - 0033 - D </td></tr><tr><td> 20mg/ml蛋白酶K溶液， </td><td> AMRESCO </td><td> E195 - 5ML </td></tr><tr><td> QIAshredder列</td><td> Qiagen公司</td><td> 79654 </td></tr><tr><td> Q解决方案</td><td> Qiagen公司</td><td>提供Taq DNA聚合酶</td></tr><tr><td>核糖核酸酶</td><td>西格玛</td><td> R6513 </td></tr><tr><td> RNase抑制剂</td><td>罗氏应用科学部</td><td> 03335399001 </td></tr><tr><td>的RNeasy微型试剂盒</td><td> Qiagen公司</td><td> 74104 </td></tr><tr><td>的RNeasy微型试剂盒</td><td> Qiagen公司</td><td> 74104 </td></tr><tr><td> RQ1 RNase的免费DNA酶</td><td> Promega公司</td><td> M6101 </td></tr><tr><td> SeaPlaque低熔化琼脂糖</td><td>龙沙</td><td> 50101 </td></tr><tr><td>羊血清</td><td>西格玛</td><td> S2263 - 500ML </td></tr><tr><td> Stericup过滤装置，0.22μm的，聚醚砜，500ML </td><td> Millipore公司</td><td> SCGPU05RE </td></tr><tr><td>叠氮化钠，呈颗粒状</td><td> Fisher Scientific则</td><td> S227I - 100 </td></tr><tr><td>氯化钠</td><td> Fisher Scientific则</td><td> BP358 - 212 </td></tr><tr><td>十二烷基硫酸钠</td><td> Fisher Scientific则</td><td> S529 - 500 </td></tr><tr><td>南南合作，20X解决方案</td><td>研究产品国际</td><td> S24022 - 4000.0 </td></tr><tr><td>标III第一链合成系统</td><td> Invitrogen公司</td><td> 18080-051 </td></tr><tr><td> T7 RNA聚合酶</td><td>罗氏应用科学部</td><td> 10881767001 </td></tr><tr><td> Taq DNA聚合酶</td><td> Qiagen公司</td><td> 201203 </td></tr><tr><td>的Tris - HCl </td><td> Fisher Scientific则</td><td> BP153 - 1 </td></tr><tr><td>吐温20 </td><td> Fisher Scientific则</td><td> BP337 - 100 </td></tr><tr><td>与豪华标本浴振动切片机</td><td>徕卡显微系统</td><td> VT1000A </td></tr><tr><td>酵母tRNA </td><td>罗氏应用科学部</td><td> 109495 </td></tr></tbody></table>

a high throughput in situ hybridization method to characterize mrna expression patterns in the fetal mouse lower urogenital tract

较低的泌尿生殖道（LUT）的发展是一个复杂的的过程。这种复杂性体现在从依赖雄激素信号和上皮-间质相互作用1,2胎儿男性尿道，前列腺的形成。了解前列腺发育的分子机制，可能揭示的增长是不恰当地从沉睡中苏醒过来，在以后的生活会引起良性前列腺增生和前列腺癌等前列腺疾病的机制。 发展LUT的解剖结构复杂。 16.5天的概念（DPC）的时间前列腺萌芽开始，许多类型的细胞存在。血管，神经和平滑肌驻留在间质的基质 3 。这间质围绕着一个多层的上皮细胞和引起胎儿通过雄激素受体 ​​依赖旁分泌信号 4前列腺。身份的基质雄激素受体反应的前列腺癌的发展，针对这些基因的前列腺导管上皮形式尚不完全清楚的机制所需基因。能够准确地识别细胞类型和本地化内表达的具体因素是要进一步了解前列腺癌的发展。原位杂交（ISH）允许一个组织内mRNA的本地化。因此，这种方法可用于识别信号分子及其受体的表达模式和时间，从而阐明潜在的前列腺发育调节。 在这里，我们描述了一种高通量的原位杂交技术来识别在胎鼠LUT的使用振动切片机切段mRNA的表达模式。这种方法提供了比其他ISH的协议几个优点。执行上坚持一个幻灯片的超薄切片的原位杂交是技术上的困难;冰冻切片经常有结构质量差而冰冻切片和石蜡切片，往往会造成弱信号的分辨率。执行整装组织的ISH，可能会导致探头捕获。相比之下，我们的高通量技术采用厚切的部分，显示详细的组织架构。修改离心管允许在原位杂交的过程中容易处理的部分。 4 mRNA转录的一个最多可从一个单一的LUT高达24 mRNA的单次运行中检测到的成绩单17.5dpc筛选，从而降低成本和最大限度地提高效率。这种方法允许多个治疗组相同，并作为一个单元处理，从而消除任何解释数据的偏差。大多数有针对性地对前列腺的研究人员，这种方法提供了一个在胎鼠尿道前列腺导管网络，使人们产生低和高丰度的mRNA转录的空间和时间的位置。

Watch this Scientific Journal Video about A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract at JoVE.com

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

在这里，我们描述了一个高效的高吞吐量<em&gt;原位</em&gt;杂交（ISH）可视化mRNA的表达模式，在胎鼠的前列腺组织切片的方法。该方法可以很容易地适应可视化，在其他的鼠标组织或来自其他物种的组织的mRNA的表达模式。

高通量原位杂交方法表征在胎鼠下泌尿生殖道的基因表达模式

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

a-high-throughput-situ-hybridization-method-to-characterize-mrna

Research

JoVE Journal

Biology

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

12.9K 次观看.  University of Wisconsin-Madison. 在这里，我们描述了一个高效的高吞吐量原位杂交（ISH）可视化mRNA的表达模式，在胎鼠的前列腺组织切片的方法。该方法可以很容易地适应可视化，在其他的鼠标组织或来自其他物种的组织的mRNA的表达模式。

视频: A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

一种高通量的方法，在全球范围内研究在酿酒酵母中的细胞器形态

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

科研

生物学

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

斑马鱼整装高分辨率双荧光原位杂交

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

一种快速高通量的方法映射核糖核蛋白（RNPs）人类的前体mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

嗨 - C：一个方法研究基因组的三维架构。

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

全山原位杂交E8.5，E11.5小鼠胚胎

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

放射性的原位杂交检测组织不同的基因表达模式

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

高通量使用自动染色体的物理图谱<em&gt;原位</em&gt;杂交

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...