这项工作得到了美国心脏协会 (P50CA150964 援助 0365274B)、国家癌症研究所 [胃肠孢子到 Z.W.]、国立卫生研究院的支持 (研究基础设施共享的仪器授权 S10RR031845位于西储大学的光镜成像设备] 和中国奖学金理事会 [X.G.]。 作者感谢理查德. 李在光镜成像核心为他的帮助与幻灯片扫描。

这里所描述的所有涉及老鼠的实验都得到了西方储备大学机构动物护理和使用委员会的批准。2016-0068 号议定书 (PI: 华娄) 的标题是 "在脊椎动物发展中替代性前基因剪接的作用"。
注: 本议定书所用的所有设备、试剂和用品的信息都列在材料表中。
1. 制备福尔马林固定石蜡嵌入 (FFPE) 切片
<ol><li>
弄死 1 CD1 鼠和 1 C57BL/6J 小鼠颈椎脱位。小心切开用手术剪刀和镊子打开头骨。用勺子把大脑移开。 注: 5 月大的 CD1 雄性小鼠和3月大的 C57BL/6J 雄性小鼠被使用并显示出相似的结果。<ol>
<li>用 PBS 洗脑。通过将整个大脑放在福尔马林溶液的50毫升内, 用含有10% 福尔马林室温 (RT) 的溶液将整个大脑固定在30小时内。</li>
		<li>改变固定的解决方案, pbs 和孵化的大脑在 pbs 过夜4摄氏度。使用标准程序18对二甲苯和石蜡进行脱水和嵌入。</li>
	</ol>
</li>
	<li>
使用切片将嵌入的组织切成5µm 的部分。首先将石蜡带放在 RT 水浴中, 然后在45摄氏度的水浴中传播丝带。
	<ol>
<li>当丝带上的皱纹和褶皱消失后, 立即在玻璃滑梯上装上部分。空气干燥幻灯片隔夜在 RT. 烘烤幻灯片为1小时在60°c 在使用之前。</li>
	</ol>
</li>
</ol>2. 样品预处理
<ol><li>
Deparaffinize 组织切片。在通风罩里执行这些步骤。
	<ol>
<li>用250毫升新鲜二甲苯和两个250毫升100% 乙醇填充两个洗涤盘 (图 1A)。将组织幻灯片插入洗涤架 (图 1A), 并将洗涤架放在洗涤盘中, 按照表 1所示的顺序和孵化时间。</li>
		<li>每次孵化期间, 在洗涤盘中向上和向下提起洗涤架3次以上。每次孵化后, 迅速将洗涤架移入下一个洗涤盘, 防止组织切片干燥。</li>
		<li>完成孵化步骤后, 从洗涤盘中取出带幻灯片的洗涤架。将机架放置在60摄氏度孵化器中5分钟, 或直到幻灯片完全干燥。</li>
	</ol>
</li>
	<li>绘制一个 0.75 "x 0.75" 屏障周围的组织部分与疏水性屏障笔。空气干燥的屏障在 RT 3 分钟。</li>
	<li>
预处理切片
	<ol>
<li>准备试剂和设备<ol>
<li>打开杂交烤箱 (图 1B), 在使用前将温度设置为40摄氏度30分钟。</li>
			<li>将加湿纸 (图 1E) 与蒸馏水完全润湿, 放入湿度控制托盘 (图 1D, 左)。将湿度控制托盘放在杂交烤箱中。</li>
			<li>700毫升的新鲜1x 目标检索试剂, 增加630毫升的 dH2O 到70毫升10x 目标检索试剂。</li>
		</ol>
</li>
		<li>应用过氧化氢。<ol>
<li>把 deparaffinized 的幻灯片放在长凳上。添加5–8滴过氧化氢到一个部分完全覆盖的组织。在 RT 孵化10分钟。</li>
			<li>点击一张吸水纸上的幻灯片以除去过氧化氢溶液, 一次滑动一次。立即将滑块插入洗涤架中, 将机架移至充满 dH2O 的洗涤盘中。</li>
			<li>通过在蒸馏水中上下移动机架3–5时间来清洗幻灯片。将洗涤架移到充满新鲜蒸馏水的洗涤盘中, 再冲洗一次。</li>
		</ol>
</li>
		<li>执行目标检索<ol>
<li>在热板上放置一个1000毫升的玻璃烧杯。在烧杯中加入700毫升新鲜的1x 目标检索试剂, 并用箔盖住。通过箔盖在烧杯中插入温度计 (图 1C)。打开热板, 设置为10–15分钟高。</li>
			<li>当检索试剂的温度达到98摄氏度时, 将热板的温度控制从高到低切换到保持轻度沸腾。不要煮沸超过15分钟。</li>
			<li>同时, 小心地打开箔, 并把洗涤架与切片内的检索摄政。再盖上烧杯, 孵化15分钟。</li>
			<li>使用镊子将机架从烧杯中移至200毫升 dH2O 的洗涤盘, 并于十五年代冲洗。</li>
			<li>将机架移到含有100% 新鲜乙醇的洗涤盘上, 让我们坐3分钟. 删除幻灯片, 并在60摄氏度孵化器中烘干10分钟。</li>
		</ol>
</li>
		<li>应用蛋白酶 III<ol>
<li>将幻灯片放在污点机架上 (图 1D, 右)。添加5滴蛋白酶 III 以覆盖组织。小心地把机架放在湿度控制托盘 (图 1D, 左), 并插入托盘到40°c 杂交烤箱。孵育30分钟。</li>
			<li>取下滑轨, 把托盘放回烤箱。点击幻灯片删除多余的液体, 一次一张幻灯片。将幻灯片插入洗衣机机架中, 并将机架放在装满 dH2的洗涤盘中, 将机架上下移动3-5 次以清洗幻灯片。</li>
		</ol>
</li>
	</ol>
</li>
</ol>3. 化验
<ol><li>
准备材料
	<ol>
<li>预热50x 洗涤缓冲器在40°c 孵化器20分钟混合2.94 升的 dH2O 和60毫升50x 洗涤缓冲器, 使1x 洗涤缓冲。</li>
		<li>用100毫升的鳃的苏木精 I 到100毫升的 dh, 制备50% 苏木素染色液2o. 将0.02% 毫升的氢氧化铵添加到1.43 毫升, 并在容器中混合, 制备 1(w/v) 氨水。</li>
		<li>使用前, 将探头和安培0–6试剂放在 RT 30 分钟。保持湿度控制托盘在40°c 杂交烤箱。</li>
	</ol>
</li>
	<li>
程序 注: 要检查Nf1拼接亚型的表达式, 并计算外显子23a 的 (PSI) 值的拼接百分比, 三种不同的探头, 目标外显子 23 a 包含异构体, 外显子 23 a 排除异构体或两个亚型均被使用 (图2).探针用于相邻切组织切片。为了保证计算的准确性, 在杂交、扩增和信号检测步骤中, 对三组切片进行完全相同的处理是至关重要的。<ol>
<li>探针杂交。<ol>
<li>从清洗机架上取出幻灯片, 然后从幻灯片中点击多余的液体。将幻灯片放在污点机架中。</li>
			<li>使用铅笔用探针名称标记幻灯片。把三相邻切脑组织幻灯片与三个不同的 Nf1 探针 (图 2) 混合在一起。按照相同的幻灯片顺序执行后续步骤。</li>
			<li>添加4滴探针覆盖组织。每次滑动一次, 以防止剖面干燥。将污渍架放在湿度控制托盘中, 并将其插入40°c 烤箱中2小时。</li>
			<li>用洗涤缓冲器清洗两次幻灯片。每次清洗时, 请用200毫升的1x 洗涤缓冲器填充染色盘, 并在其中放置一个洗涤架。一次性在纸巾上轻拍多余的液体, 然后迅速将其插入洗涤架中。将幻灯片架向上和向下移动到盘中, 3–5时间为2分钟的 RT。</li>
			<li>第二次清洗时, 使用新鲜的洗涤缓冲器。</li>
		</ol>
</li>
		<li>信号放大。<ol>
<li>从幻灯片中点击多余的洗涤缓冲器, 将它们放在污渍架中。添加4滴安培 0, 以覆盖组织。将污渍架放在湿度控制托盘中, 将其插入烤箱中30分钟, 40 摄氏度。</li>
			<li>洗两次幻灯片的洗涤缓冲, 如上文所述。</li>
			<li>按照表 2的顺序和条件重复此步骤与 AMP 1–6。每次杂交步骤后两次清洗幻灯片。</li>
		</ol>
</li>
		<li>信号检测<ol>
<li>从幻灯片上点击多余的洗涤缓冲器, 将它们放在污渍架上。在离心管中添加2µL 的快速红 B 到120µL 的快速红色 A (1:60 比值)。混合好, 并添加到完全覆盖的组织部分。 注: 红/乙混合料的总容积是根据组织切片的数量和大小而计算的。对于 0.75 "x 0.75" 区域, 120 µL 足够用于一个部分。使用5分钟内的混合, 避免暴露的混合, 以直接阳光或 UV 光。</li>
			<li>关闭托盘和孵化10分钟的 RT. 将解决方案移至废物容器, 并立即将其插入洗涤架中, 并将机架放在装满自来水的洗涤盘中。再用新鲜自来水冲洗。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
Counterstaining 幻灯片
	<ol>
<li>从自来水中取出洗涤架, 然后快速地用吸水纸来除去多余的水。将机架放入50% 苏木精染色溶液中, 并立即将机架上下移动几次。在 RT 孵化2分钟。</li>
		<li>将滑片架移回自来水盘中。清洗3–5时间通过移动机架上下和改变新鲜自来水, 直到幻灯片变得清晰, 而脑组织保持紫色。</li>
		<li>将机架移至含有0.02% 氨水的盘子中。将机架向上和向下移动2–3时间, 直到组织变成蓝色。用新鲜的自来水在染色盘中3–5时间冲洗滑梯。</li>
	</ol>
</li>
	<li>
样品的安装
	<ol>
<li>将滑块从染色盘移动到60摄氏度孵化器, 并至少孵化15分钟, 直到幻灯片完全干燥。</li>
		<li>放置40µL 的安装介质在幻灯片上, 并小心地放置一个24毫米 x 50 毫米盖玻片在组织部分。空气干燥幻灯片在 RT 30 分钟。</li>
	</ol>
</li>
</ol>4. 数据收集和分析
注意: 使用幻灯片扫描仪以40X 倍的比例扫描图像。
<ol><li>
正和负控制
	<ol>
<li>对于每个实验, 包括正负控制 (图 3)。作为一个良好的正则控制, 信号应该是可见的, 在20-40X 放大倍数点。</li>
		<li>使用鼠标 (Mm)-PPIB 1 zz 作为一个积极的控制。这个探针的目标是一个常见的管家基因 PPIB。对于负控制, 每个20细胞显示每20X 显微镜场背景染色的一个点是可以接受的。</li>
		<li>使用 DapB-1ZZ 探头作为负控制。该探针针对细菌基因 dapB。这些控制探头已用于许多19。</li>
	</ol>
</li>
	<li>
Nf1外显子23a 的信号检测与表达模式分析
	<ol>
<li>当杂交信号被表示为有斑点点时, 手动计数点。点数与特定探针检测到的成绩单的水平相关。注意, 点数也可以通过 ImageJ 或 SpotStudio 软件进行分析。</li>
		<li>使用三探针杂交至外显子 23 a 包含, 外显子 23 a 缺乏亚型, 或总Nf1记录 (图 2)。由于三探头不能同时使用, 使用三相邻的脑组织切片杂交到每个探头。<ol>
<li>要计算外显子23a 的拼接百分比 (PSI), 请计算数个脑区中的细胞数和圆点个数。对于每个感兴趣的区域, 按图 4所示, 从多个子区域中计数超过400个单元格。</li>
			<li>收集来自同一子区域的三个探测器的数据。将 PSI 计算为每个单元格的点数与外显子23a 包含探针/(每个单元格的点数与外显子23a 包含探针 + 每个单元格的点数与 exon23a 跳过探针) x 100%。</li>
		</ol>
</li>
	</ol>
</li>
</ol>

<ol>
	<li>Wang, E. T., 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=Alternative+isoform+regulation+in+human+tissue+transcriptomes.">Alternative isoform regulation in human tissue transcriptomes.</a> Nature. 456, 470-476 (2008).</li><li>Pan, Q., Shai, O., Lee, L. J., Frey, B. J., Blencowe, 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=Deep+surveying+of+alternative+splicing+complexity+in+the+human+transcriptome+by+high-throughput+sequencing.">Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing.</a> Nature Genetics. 40, 1413-1415 (2008).</li><li>Cieply, B., Carstens, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+roles+of+alternative+splicing+factors+in+human+disease.">Functional roles of alternative splicing factors in human disease.</a> Wiley Interdisciplinary Reviews: RNA. 6, 311-326 (2015).</li><li>Xiong, H. 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=RNA+splicing.+The+human+splicing+code+reveals+new+insights+into+the+genetic+determinants+of+disease.">RNA splicing. The human splicing code reveals new insights into the genetic determinants of disease.</a> Science. 347, 1254806 (2015).</li><li>Shirai, Y., Watanabe, M., Sakagami, H., Suzuki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+splice+variants+in+the+5'UTR+of+Gtf2i+expressed+in+the+rat+brain:+alternative+5'UTRs+and+differential+expression+in+the+neuronal+dendrites.">Novel splice variants in the 5'UTR of Gtf2i expressed in the rat brain: alternative 5'UTRs and differential expression in the neuronal dendrites.</a> Journal of Neurochemistry. 134, 578-589 (2015).</li><li>Cui, Y., Liu, J., Irudayaraj, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+quantification:+in+situ+analysis+of+transcriptome+and+pre-mRNA+alternative+splicing+at+the+nanoscale.">Beyond quantification: in situ analysis of transcriptome and pre-mRNA alternative splicing at the nanoscale.</a> Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology. 9, (2017).</li><li>Trifonov, S., Yamashita, Y., Kase, M., Maruyama, M., Sugimoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamic+acid+decarboxylase+1+alternative+splicing+isoforms:+characterization,+expression+and+quantification+in+the+mouse+brain.">Glutamic acid decarboxylase 1 alternative splicing isoforms: characterization, expression and quantification in the mouse brain.</a> BMC Neuroscience. 15, 114 (2014).</li><li>Hollander, D., Naftelberg, S., Lev-Maor, G., Kornblihtt, A. R., Ast, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Are+Short+Exons+Flanked+by+Long+Introns+Defined+and+Committed+to+Splicing?.">How Are Short Exons Flanked by Long Introns Defined and Committed to Splicing?.</a> Trends in Genetics. 32, 596-606 (2016).</li><li>Cassidy, A., Jones, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+in+in+situ+hybridisation.">Developments in in situ hybridisation.</a> Methods. 70, 39-45 (2014).</li><li>Meshorer, 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=Alternative+splicing+and+neuritic+mRNA+translocation+under+long-term+neuronal+hypersensitivity.">Alternative splicing and neuritic mRNA translocation under long-term neuronal hypersensitivity.</a> Science. 295, 508-512 (2002).</li><li>Wang, F., 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=RNAscope:+a+novel+in+situ+RNA+analysis+platform+for+formalin-fixed,+paraffin-embedded+tissues.">RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.</a> Journal of Molecular Diagnostics. 14, 22-29 (2012).</li><li>Wang, 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=RNAscope+for+in+situ+detection+of+transcriptionally+active+human+papillomavirus+in+head+and+neck+squamous+cell+carcinoma.">RNAscope for in situ detection of transcriptionally active human papillomavirus in head and neck squamous cell carcinoma.</a> Journal of Visualized Experiments. , (2014).</li><li>Zhu, H., Hinman, M. N., Hasman, R. A., Mehta, P., Lou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+neuron-specific+alternative+splicing+of+neurofibromatosis+type+1+pre-mRNA.">Regulation of neuron-specific alternative splicing of neurofibromatosis type 1 pre-mRNA.</a> Molecular and Cellular Biology. 28, 1240-1251 (2008).</li><li>Hinman, M. N., Sharma, A., Luo, G., Lou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurofibromatosis+type+1+alternative+splicing+is+a+key+regulator+of+Ras+signaling+in+neurons.">Neurofibromatosis type 1 alternative splicing is a key regulator of Ras signaling in neurons.</a> Molecular and Cellular Biology. 34, 2188-2197 (2014).</li><li>Nguyen, H. T., 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=Neurofibromatosis+type+1+alternative+splicing+is+a+key+regulator+of+Ras/ERK+signaling+and+learning+behaviors+in+mice.">Neurofibromatosis type 1 alternative splicing is a key regulator of Ras/ERK signaling and learning behaviors in mice.</a> Human Molecular Genetics. 26, 3797-3807 (2017).</li><li>Zhou, H. 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=Hu+proteins+regulate+alternative+splicing+by+inducing+localized+histone+hyperacetylation+in+an+RNA-dependent+manner.">Hu proteins regulate alternative splicing by inducing localized histone hyperacetylation in an RNA-dependent manner.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, E627-E635 (2011).</li><li>Sharma, 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=Calcium-mediated+histone+modifications+regulate+alternative+splicing+in+cardiomyocytes.">Calcium-mediated histone modifications regulate alternative splicing in cardiomyocytes.</a> Proceedings of the National Academy of Sciences of the United States of America. 111, E4920-E4928 (2014).</li><li>Adzemovic, M. Z., 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=Immunohistochemical+Analysis+in+the+Rat+Central+Nervous+System+and+Peripheral+Lymph+Node+Tissue+Sections.">Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections.</a> Journal of Visualized Experiments. , (2016).</li><li>Shokry, I. M., Callanan, J. J., Sousa, J., Tao, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+In+Situ+Hybridization+using+Oligonucleotide+Probes+on+Paraformaldehyde-prefixed+Brain+of+Rats+with+Serotonin+Syndrome.">Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome.</a> Journal of Visualized Experiments. , (2015).</li><li>Erben, L., He, M. X., Laeremans, A., Park, E., Buonanno, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Ultrasensitive+In+Situ+Hybridization+Approach+to+Detect+Short+Sequences+and+Splice+Variants+with+Cellular+Resolution.">A Novel Ultrasensitive In Situ Hybridization Approach to Detect Short Sequences and Splice Variants with Cellular Resolution.</a> Molecular Neurobiology. , (2017).</li></ol>

作者没有什么可透露的。

这项通信报告的使用 BaseScope RNA, 以检查作为表达模式的小鼠脑科。结果表明, 小于50核苷酸的反感外显子-显子结探针能更准确地靶向外显子夹杂和跳过亚型。此外, 由此产生的信号可以用来计算一个替代外显子的 PSI。
在程序中测试了一些变体。例如, 通过恒温器切片生成的冰冻组织切片进行了测试, 并证明其在该协议中的应用是成功的。然而, 在这种情况下, deparaffinization 步骤在2.1 被改变为洗涤 OCT 与 PBS 5 分钟。此外, 虽然使用由加法器提供的杂交烤箱是非常方便的, 使用一个简单的孵化器设置在40°c 结合一个滑动染色托盘用于免疫组化提供了可比的结果。重要的是要保持在盒子里的水分, 包括湿滤纸在整个过程中。
这个程序有一个限制。目前, 在同一组织幻灯片上执行多色标签是不可行的。因此, 不同的探头只能用于相邻切割, 不同的部分。为了获得准确的 PSI 值, 必须确保探针杂交的潜伏期、信号放大和检测对每个组织切片都是相同的。在未来, 使用多色探针在同一组织幻灯片, 如使用的 RNAscope 程序, 将是非常可取的。
本报告中所描述的 RNA, 提供了一种强有力的新工具来定量地分析作为原位的表达模式。该技术可用于研究多种替代外显子。在最近的一份报告中, 它成功地用于研究四 ErbB4 剪接亚型在细胞20的神经元和少突胶质中的差异表达模式。如这和其他几份报告所示, BaseScope RNA 与免疫化学的结合将是研究差异表达剪接亚型6、20的定位和功能的有力方法。

替代剪接 (AS) 是一个常见的过程中发生的前 mRNA 成熟。在这个过程中, 一个外显子可以是差异包括在成熟的 mRNA。因此, 通过作为, 一个基因可以产生许多基因编码为不同的蛋白质产品。据估计, 人类基因的92–94% 可替代剪接1,2。基因突变导致的不正常的选择性剪接模式与大量疾病有关, 包括肌萎缩侧索硬化、强直营养不良和癌症3、4。因此, 调查和更好地了解替代剪接调控机制, 以寻求新的人类疾病治疗, 是至关重要的。
通常是以特定于细胞类型的方式来调节的。确定特定基因在给定生物系统中的表达模式是很重要的。然而, 当一个包含许多不同类型细胞的复杂器官 (如大脑或心脏) 被研究时, 这就变得复杂了。在这种情况下, 一个理想的检测系统的选择是 RNA原位杂交 (用组织切片), 因此, 作为特定基因的表达模式可以在许多细胞类型同时检测。事实上, 外显子特定的探针被用来评估替代外显子5,6,7的表达水平。但是, 由于以下原因, 这种方法不太适合作为模式分析。首先, 常规的方法通常使用比 300 bp 长的探针, 而脊椎动物内部外显子 (不是第一个或最后一个外显子) 的平均大小是170核苷酸8,9。其次, 当一个外显子特定探针用于检查内部替代外显子的剪接模式时, 探针检测到的唯一的 mrna 异构体是含有外显子的, 而无外显子的 mrna 异构体无法检测到。因此, 计算替代外显子的 (PSI) 值的拼接百分比是复杂的。此外, 传统的荧光染色通常与多相结合, 降低了检测效率和鲁棒性。例如, 在研究了乙酰胆碱酯酶 (疼痛) mRNA 的应力诱导剪接异构体切换时, 辛被纳入到探头中, 并使用抗辛抗体进行检测。或者, 用碱性磷酸酶/链亲和素共轭和碱性磷酸酶10的基质检测生物素标记探针。两种方法都不使用任何放大策略来提高检测的灵敏度。因此, 检测在低水平表达的 mRNA 转录是很有挑战性的。因此, 需要一个更简单、更健壮的检测系统来分析原位的表达模式。
BaseScope 是在 RNAscope 的基础上发展起来的, 该平台是一个建立良好、应用广泛的检测系统。两种检测系统都采用目标特定的放大技术, 提高了检测11、12的灵敏度。区别于另一种的是目标序列的长度, 这是 BaseScope 的50核苷酸, 300–1,000 核苷酸用于 RNAscope。因此, 有可能设计的探针, 目标外显子-显子的连接, 以检测特定的替代 mRNA 亚型。在目前的研究中, 建立了一个程序, 以检查神经纤维瘤1型 (Nf1) 外显子23a 的表达模式, 在同一实验室13,14,15广泛研究的替代显子。,16,17、在小鼠脑科。结果表明, BaseScope 是研究Nf1外显子 23a原位表达模式的理想系统。由于该检测系统可以被改编为许多替代外显子的表达模式, 它代表了一个强大的新工具在研究中的作为。

<table>
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Oven</td>
			<td>Advanced Cell Diagnostics</td>
			<td>241000ACD</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidity Control Tray (with lid)</td>
			<td>Advanced Cell Diagnostics</td>
			<td>310012</td>
			<td></td>
		</tr>
		<tr>
			<td>Stain Rack</td>
			<td>Advanced Cell Diagnostics</td>
			<td>310017</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Fisher Scientific</td>
			<td>1160049SH</td>
			<td></td>
		</tr>
		<tr>
			<td>Imperial III General Purpose Incubator</td>
			<td>Lab-Line</td>
			<td>302</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide Scanner</td>
			<td>Leica</td>
			<td>SCN400</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pretreatment kit</td>
			<td>Advanced Cell Diagnostics</td>
			<td>322381</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide</td>
			<td>Advanced Cell Diagnostics</td>
			<td>2000899</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease III*</td>
			<td>Advanced Cell Diagnostics</td>
			<td>2000901</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Target Retrieval</td>
			<td>Advanced Cell Diagnostics</td>
			<td>2002555</td>
			<td></td>
		</tr>
		<tr>
			<td>BaseScope Detection Reagent Kit</td>
			<td>Advanced Cell Diagnostics</td>
			<td>332910</td>
			<td></td>
		</tr>
		<tr>
			<td>AMP 0</td>
			<td>Advanced Cell Diagnostics</td>
			<td>2001814</td>
			<td></td>
		</tr>
		<tr>
			<td>AMP 1</td>
			<td>Advanced Cell Diagnostics</td>
			<td>2001815</td>
			<td></td>
		</tr>
		<tr>
			<td>AMP 2</td>
			<td>Advanced Cell Diagnostics</td>
			<td>2001816</td>
			<td></td>
		</tr>
		<tr>
			<td>AMP 3</td>
			<td>Advanced Cell Diagnostics</td>
			<td>2001817</td>
			<td></td>
		</tr>
		<tr>
			<td>AMP 4</td>
			<td>Advanced Cell Diagnostics</td>
			<td>16229B</td>
			<td></td>
		</tr>
		<tr>
			<td>AMP 5-RED</td>
			<td>Advanced Cell Diagnostics</td>
			<td>16229C</td>
			<td></td>
		</tr>
		<tr>
			<td>AMP 6-RED</td>
			<td>Advanced Cell Diagnostics</td>
			<td>2001820</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast RED-A</td>
			<td>Advanced Cell Diagnostics</td>
			<td>2001821</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast RED-B</td>
			<td>Advanced Cell Diagnostics</td>
			<td>16230F</td>
			<td></td>
		</tr>
		<tr>
			<td>50X Wash Buffer</td>
			<td>Advanced Cell Diagnostics</td>
			<td>310091</td>
			<td></td>
		</tr>
		<tr>
			<td>Negative Control Probe- Mouse DapB-1ZZ</td>
			<td>Advanced Cell Diagnostics</td>
			<td>701021</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive Control Probe- Mouse (Mm)-PPIB-1ZZ</td>
			<td>Advanced Cell Diagnostics</td>
			<td>701081</td>
			<td></td>
		</tr>
		<tr>
			<td>Control slide-mouse 3T3 cell pellet</td>
			<td>Advanced Cell Diagnostics</td>
			<td>310045</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Other supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Humidifying Paper</td>
			<td>Advanced Cell Diagnostics</td>
			<td>310015</td>
			<td></td>
		</tr>
		<tr>
			<td>Washing Rack</td>
			<td>American Master Tech Scientific</td>
			<td>9837976</td>
			<td></td>
		</tr>
		<tr>
			<td>Washing Dishes</td>
			<td>American Master Tech Scientific</td>
			<td>LWS20WH</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophobic Barrier Pen</td>
			<td>Vector Laboratory</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Glass, 24 x 50 mm</td>
			<td>Fisher Scientific</td>
			<td>12--545-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>002689</td>
			<td></td>
		</tr>
		<tr>
			<td>100% ethanol (EtOH)</td>
			<td>Decon Labs</td>
			<td>2805</td>
			<td></td>
		</tr>
		<tr>
			<td>formalde solution</td>
			<td>Fisher Scientific</td>
			<td>SF94-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin I</td>
			<td>American Master Tech Scientific</td>
			<td>17012359</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Medium</td>
			<td>Vector Labs</td>
			<td>H-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Fisher Scientific</td>
			<td>173942</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative analysis of alternative pre-mrna splicing in mouse brain sections using rna in situ hybridization assay

替代剪接 (AS) 发生在超过90% 的人类基因。一个交替拼接的外显子的表达模式通常是以特定于细胞类型的方式来调节的。由于表达模式通常由 RT PCR 和 rna 序列分析, 使用 rna 样本从细胞的数量分离。原位检测作为一种特定的生物结构的表达模式, 可以通过 RNA原位杂交 (用外显子特异探针) 进行。然而, 这种特殊用途的使用是有限的, 因为替代外显子通常太短, 以设计显子特定的探针。在本报告中, BaseScope 的使用, 最近开发的技术, 采用短反义寡核苷酸在 RNA, 被描述为分析模式在小鼠脑科。用神经纤维瘤1型 (Nf1) 的外显子23a 作为例, 说明短外显子-显像结合探针在小鼠脑切片 RNA 分析中表现出高特异性的鲁棒杂交信号。更重要的是, 用外显子包含和跳过特定探针检测到的信号可以用来可靠地计算在小鼠大脑不同解剖区域Nf1外显子23a 表达式的值拼接的百分比。给出了分析的实验协议和计算方法。结果表明, BaseScope 提供了一个强大的新的工具来评估作为表达模式的原位。

BaseScope 使用三小鼠菌株: CD1 野生型小鼠, C57BL/6J 野生型小鼠, Nf123/23 , C57BL/6J 背景下的突变小鼠, 其中外显子23a 被包括在所有细胞类型, 由于工程剪接网站突变14,15。
作为第一步, 测试系统使用公司提供的试剂进行检测: 有3T3 细胞的幻灯片, 以及阴性和阳性的探针。如图 3所示, 当使用负控制探头时, 没有检测到信号, 而使用正控探针时检测到许多有斑点的点。
接下来, 使用鼠标大脑部分和Nf1特定的探头进行检测, 这些探针设计用于探测包含外显子23a、跳过显子23a 或两种亚型的Nf1记录 (图 2)。这些探针的目标是特定的外显子。选择两个脑区来检查Nf1外显子23a 的表达模式: 皮质和海马 CA3 (图 4)。对于每个区域, 如图 4所示, 计数三个子区域, 同时记录单元格和点号。对于每个区域, 大约有400个细胞被计算在总数中, 用于计算点/细胞比。作为Nf1外显子23a 的表达式模式被计算为 PSI。
图 5A-5F和表 3显示了三Nf1探针在 CD1 小鼠皮层和海马中获得的信号。还分析了 C57BL/6J Nf1 和 Nf123-23 小鼠的皮质区 (图 6)。
这些实验的结果导致了几个结论。首先, 显示为点/单元格的信号的总和, 由外显子23a 包含特定的和跳过特定的探头检测, 等于探测器检测到的混合到两种亚型 (表 3), 这表明, 三探针杂交与Nf1成绩单具有类似的效率。第二, 10% 的皮层外显子23a 的 PSI 值与先前报告的 rt-pcr 结果一致, 其中皮质从成年 C57BL/6J 小鼠脑中解剖, 其次是总 RNA 分离和 rt-pcr 分析14,15。这一结果表明, BaseScope 可用于定量分析作为表达模式, 除了转录水平的组织切片。第三, 结果与Nf123/23 的突变脑组织, 其中所有的Nf1记录包含外显子 23a14,15, 显示类似水平的信号时, 包含特定的和使用Nf1总探头, 并且在使用跳过特定探头时没有信号 (图 6), 这表明两个探针靶向外显子包含或跳跃是高度特异的。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57889/57889fig1.jpg"> 图 1: 使用的设备.(A) 洗涤盘和洗涤架。(B) 杂交烤箱。(C) 目标检索烧杯和热板集。(D) 湿度控制托盘和污渍架。(E) 湿度控制托盘内红色箭头指示的加湿纸。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57889/57889fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57889/57889fig2.jpg"> 图 2: 用于检测Nf1外显子23a 作为表达式模式的探头.所有探头都包含一条 ZZ 对寡核苷酸。包含特定探针 (red) 针对外显子23a 和24的连接点, 跳过特定探针 (绿色) 针对外显子23和24的连接, 而Nf1总探针 (蓝色) 针对外显子26和27的连接。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57889/57889fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57889/57889fig3.jpg"> 图 3:对3T3 细胞使用阴性 (a B) 和阳性 (C D) 控制。在协议中, 通过步骤 2 (样品预处理) 和 3 (程序) 来处理包含3T3 单元格的控制幻灯片。正向信号显示为红色箭头指示的红色斑点点。B 和 D 分别放大 A 和 C 的图像。刻度条 = 20 µm (A 和 C);5µm (B 和 D)。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57889/57889fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57889/57889fig4.jpg"> 图 4: 鼠标大脑冠状部分的图片.红色方框显示了为计数细胞和信号数字在皮层和海马 CA3 区域选择的区域。刻度条 = 1 毫米.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57889/57889fig4large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57889/57889fig5.jpg"> 图 5: 使用Nf1特定探针和小鼠脑切片检测到的 RNA 信号。CD1 的大脑是通过协议中的步骤1-4 进行处理的。当使用三Nf1特定探头 (如图 3所示) 时, 红色有斑点的点表示正信号。皮质 (C) 和海马 (D F) 显示。Nf1 总探针用于 a 和 D, 包含特定探针在 B 和 E, 并且跳过特定探针在 C 和 f 刻度条 = 40 µm (A C);400µm (D F)。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57889/57889fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57889/57889fig6.jpg"> 图 6: Nf1 中检测到的 RNA 信号, Nf123/23 脑部.两个小鼠菌株都在 C57BL/6J 背景下。大脑是通过协议中的步骤1-4 进行处理的。A-C。Nf1 皮质。D F。Nf123/23 的皮质。Nf1 总探针用于 B 和 E 中的 A 和 D, 包含特定探针, 和跳过特定探针在 C 和 f 刻度杆 = 40 µm.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57889/57889fig6large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>孵化顺序</td>
			<td>TT 菜中的化学物质</td>
			<td>时间</td>
		</tr>
<tr>
<td>1</td>
			<td>250毫升二甲苯</td>
			<td>5分钟</td>
		</tr>
<tr>
<td>2</td>
			<td>250毫升二甲苯</td>
			<td>5分钟</td>
		</tr>
<tr>
<td>3</td>
			<td>250毫升100% 乙醇</td>
			<td>2分钟</td>
		</tr>
<tr>
<td>4</td>
			<td>250毫升100% 乙醇</td>
			<td>2分钟</td>
		</tr>
</tbody></table>表 1:步骤2.1.1 的孵化过程.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>安培解决方案</td>
			<td>孵化时间</td>
			<td>孵化温度</td>
		</tr>
<tr>
<td>安培1</td>
			<td>15分钟</td>
			<td>40°c</td>
		</tr>
<tr>
<td>安培2</td>
			<td>30分钟</td>
			<td>40°c</td>
		</tr>
<tr>
<td>安培3</td>
			<td>30分钟</td>
			<td>40°c</td>
		</tr>
<tr>
<td>安培4</td>
			<td>15分钟</td>
			<td>40°c</td>
		</tr>
<tr>
<td>安培 5-红色</td>
			<td>30分钟</td>
			<td>室温</td>
		</tr>
<tr>
<td>安培 6-红色</td>
			<td>15分钟</td>
			<td>室温</td>
		</tr>
</tbody></table>表 2:步进3.2.2 信号放大程序.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>地区</td>
			<td>跳</td>
			<td>包含</td>
			<td>跳过 + 包含</td>
			<td>总</td>
			<td>Psi</td>
		</tr>
<tr>
<td>皮质</td>
			<td>2.9±0。1</td>
			<td>0.32±0.03</td>
			<td>3.21</td>
			<td>3.22</td>
			<td>10</td>
		</tr>
<tr>
<td>CA3</td>
			<td>5.37±0。2</td>
			<td>0.2±0.04</td>
			<td>5.56</td>
			<td>5.65</td>
			<td>3.71</td>
		</tr>
</tbody></table>表 3:从图 5所示的图像中计算出 RNA 的结果.数字 (以点/单元格表示) 是使用400多个细胞计算的, 它们包括在皮质和 CA3 的三个子区域 (图 4)。包括标准错误。

Watch this Scientific Journal Video about Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay at JoVE.com

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

本文介绍了一种利用短反义寡核苷酸检测小鼠脑部替代前 mRNA 拼接模式的原位杂交协议。

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 ...

quantitative-analysis-alternative-pre-mrna-splicing-mouse-brain

Nanyang Technological University

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Genetics

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

Unit 1

Transcription: DNA to RNA

SCIENTISTS IN ACTION

8.8K 次观看.  Case Western Reserve University. 本文介绍了一种利用短反义寡核苷酸检测小鼠脑部替代前 mRNA 拼接模式的原位杂交协议。

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

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

工程人为因素来特异性地操纵人细胞中的选择性剪接

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

科研

遗传学

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，<em&gt;白腹皮蠹</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these diseases, do not exist in isolation when they colonize the vector; rather, they likely engage in interactions with resident microorganisms in the gut lumen. The vector microbiota has been demonstrated to play an important role in pathogen transmission for several vector-borne diseases. Whether resident bacteria in the gut of the Ixodes scapularis tick, the vector of several human pathogens including Borrelia burgdorferi, influence tick transmission of pathogens is not determined. We require methods for characterizing the composition of the bacteria associated with the tick gut to facilitate a better understanding of potential interspecies interactions in the tick gut. Using whole-mount in situ hybridization to visualize RNA transcripts associated with particular bacterial species allows for the collection of qualitative data regarding the abundance and distribution of the microbiota in intact tissue. This technique can be used to examine changes in the gut microbiota milieu over the course of tick feeding and can also be applied to analyze expression of tick genes. Staining of whole tick guts yield information about the gross spatial distribution of target RNA in the tissue without the need for three-dimensional reconstruction and is less affected by environmental contamination, which often confounds the sequencing-based methods frequently used to study complex microbial communities. Overall, this technique is a valuable tool that can be used to better understand vector-pathogen-microbiota interactions and their role in disease transmission.

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Here, we present a protocol to spatially and temporally assess the presence of viable microbiota in tick guts using a modified whole-mount in situ hybridization approach.

全挂载原位杂交法可视化蜱内脏微生物群

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

单分子荧光原位杂交 (smFISH) 在发芽酵母营养生长和减数分裂中的分析

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To date, miRNAs have been implicated in a large number of biological and disease processes, which has signified the need for the reliable detection methods of miRNA transcripts. Here, we describe a detailed protocol for digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe-based miRNA detection, combined with protein immunostaining on mouse heart sections. First, we performed an in situ hybridization technique using the probe to identify miRNA-182 expression in heart sections from control and cardiac hypertrophy mice. Next, we performed immunostaining for cardiac Troponin T (cTnT) protein, on the same sections, to co-localize miRNA-182 with the cardiomyocyte cells. Using this protocol, we were able to detect miRNA-182 through an alkaline phosphatase based colorimetric assay, and cTnT through fluorescent staining. This protocol can be used to detect the expression of any miRNA of interest through DIG-labeled LNA probes, and relevant protein expression on mouse heart tissue sections.

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.

原位杂交法在小鼠心脏切片组织特异 miRNA 表达谱分析中的应用

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

利用体外和细胞内形状研究小分子诱导的预 mrna 结构变化

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.

单分子荧光原位杂交 (SM-FISH) 在小鼠卵母细胞中 Mrna 的定量和定位

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), ...

Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis

Human papillomavirus (HPV) infection is a major risk factor for a subtype of oropharyngeal squamous cell carcinoma (OPSCC), which tends to be associated with a better outcome than alcohol- and tobacco-related OPSCC. Chromogenic in situ hybridization (CISH) of HPV viral RNA could allow the semiquantitative evaluation of viral transcripts of the oncogenic proteins E6 and E7 and an in situ visualization with a good spatial resolution. This technique allows the diagnosis of an active infection with the visualization of HPV transcription in the tumoral HPV-infected cells. An advantage of this technique is the avoidance of contamination from nonneoplastic HPV-infected cells adjacent to the tumor. Overall, its good diagnosis performances have it considered to be the gold standard for active HPV infection identification. Since E6 and E7 viral protein interaction with cell proteins pRb and p53 is mandatory for cell transformation, HPV RNA CISH is functionally relevant and acutely reflects active oncogenic HPV infection. This technique is clinically relevant as well since &#34;low&#34; or &#34;high&#34; HPV transcription levels helped the identification of two prognosis groups among HPV-related p16-positive head and neck cancer patients. Here we present the protocol for manual HPV RNA CISH performed on formalin-fixed paraffin-embedded (FFPE) slides with a kit obtained from the manufacturer. Instead of chromogenic revelation, RNA in situ hybridization may also be performed with fluorescent revelation (RNA FISH). It may also be combined with conventional immunostaining.

Human papillomavirus (HPV) RNA chromogenic in situ hybridization is considered to be one of the gold standards for active human papillomavirus infection detection within tumors. It allows the visualization of HPV E6-E7 mRNA expression with localization and semiquantitative evaluation of its signal.

原位杂交的色原性杂交是HPV相关头颈部癌症诊断的工具

Human papillomavirus (HPV) infection is a major risk factor for a subtype of oropharyngeal squamous cell carcinoma (OPSCC), which tends to be associated ...

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and especially methylations, has re-emerged due to the important roles played by the enzymes that write and erase them in biological processes relevant to disease and cancer. Here, a sensitive in vitro assay for accurate analysis of RNA methylation writer activity on synthetic or in vitro transcribed RNAs is provided. This assay uses a tritiated form of S-adenosyl-methionine, resulting in direct labeling of methylated RNA with tritium. The low energy of tritium radiation makes the method safe, and pre-existing methods of tritium signal amplification, make it possible to quantify and to visualize the methylated RNA without the use of antibodies, which are commonly prone to artifacts. While this method is written for RNA methylation, few tweaks make it applicable to the study of other RNA modifications that can be radioactively labeled, such as RNA acetylation with 14C acetyl coenzyme A. Overall, this assay allows to quickly assess RNA methylation conditions, inhibition with small molecule inhibitors, or the effect of RNA or enzyme mutants, and provides a powerful tool to validate and expand results obtained in cells.

Here, an antibody-free in vitro assay for direct analysis of methyltransferase activity on synthetic or in vitro transcribed RNA is described.