Name: quantitative analysis of alternative pre-mrna splicing in mouse brain sections using rna in situ hybridization assay
Uploaded: 2018-08-26T13:00:00.000+00:00
Duration: 11 min 22 s
Description: 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

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>

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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...

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>&lt;strong&gt;Equipment&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Pretreatment kit&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Other supplies&lt;/strong&gt;</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 mm x 50 mm</td><td>Fisher Scientific</td><td>12--545-F</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</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<st...

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 ...

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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

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 nucleotides apart result in either the inclusion (S) or exclusion (&#916;S) of a single residue, Serine-701. Further downstream, splicing at a pair of alternative acceptor splice sites result in transcripts encoding either the 55 terminal residues of the transactivation domain (&#945;) or a truncated transactivation domain with 7 unique residues (&#946;). As outlined in this manuscript, measuring the proportions of STAT3's four spliced transcripts (S&#945;, S&#946;, &#916;S&#945; and &#916;S&#946;) was possible using absolute qPCR (quantitative polymerase chain reaction). The protocol therefore distinguishes and measures highly similar splice variants. Absolute qPCR makes use of calibrator plasmids and thus specificity of detection is not compromised for the sake of efficiency. The protocol necessitates primer validation and optimization of cycling parameters. A combination of absolute qPCR and efficiency-dependent relative qPCR of total STAT3 transcripts allowed a description of the fluctuations of STAT3 splice variants' levels in eosinophils treated with cytokines. The protocol also provided evidence of a co-splicing interdependence between the two STAT3 splicing events. The strategy based on a combination of the two qPCR techniques should be readily adaptable to investigation of co-splicing at other tandem splicing sites.

Tandem splicing events occur at sites less than 12 nucleotides apart. Quantifying ratios of such splice variants is feasible using an absolute quantitative PCR approach. This manuscript describes how splice variants of the gene STAT3, in which two splicing events results in Serine-701 inclusion/exclusion and &#945;/&#946; C-termini, can be quantified.

合并绝对和相对定量PCR数据来量化STAT3剪接变体成绩单

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 ...

科研

遗传学

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

利用RNA测序来检测新剪接变异体在相关耐药<em&gt;体外</em&gt;癌症模型

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

癌症研究

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

双荧光原位杂交食脑切片

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

神经科学

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), ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

在脑切片检测轴突本地化mRNAs的高空间分辨率<em&gt;原位</em&gt;杂交

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

结合双荧光<em&gt;原位</em&gt;杂交免疫标记为三个基因在小鼠大脑切片中表达的检测

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and &#947;-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

检测选择性剪接的过程中上皮 - 间质转化

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various ...

生物学

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust technologies that measure RNA biomarkers in situ in clinical specimens. While grind-and-bind assays like RNAseq and quantitative RT-PCR enable highly sensitive gene expression measurements, they also require RNA extraction and thus prevent valuable expression analysis within the morphological tissue context. The in situ hybridization (ISH) assay described here can detect RNA target sequences as short as 50 nucleotides at single-nucleotide resolution and at the single-cell level. This assay is complementary to the previously developed commercial assay and enables sensitive and specific in situ detection of splice variants, short targets, and point mutations within the tissue. In this protocol, probes were designed to target unique exon junctions for two clinically important splice variants, EGFRvIII and MET&#916;14. The detection of short target sequences was demonstrated by the specific detection of CDR3 sequences of T-cell receptors &#945; and &#946; in the Jurkat T-cell line. Also shown is the utility of this ISH assay for the distinction of RNA target sequences at single-nucleotide resolution (point mutations) through the visualization of EGFR L858R and KRAS G12A single-nucleotide variations in cell lines using automated staining platforms. In summary, the protocol shows a specialized RNA ISH assay that enables the detection of splice variants, short sequences, and mutations in situ for manual performance and on automated stainers.

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Here, we describe an in situ hybridization assay which enables sensitive and specific detection of sequences as short as 50 nucleotides with single-nucleotide resolution at the single-cell level. The assay, which can be performed manually or automatically, can enable visualization of splice variants, short sequences, and mutations within the tissue context.

用 RNA原位杂交法对形态学组织环境中的遗传变异、短靶点和突变进行可视化

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust ...

Identification of Alternative Splicing and Polyadenylation in RNA-seq Data

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also be utilized to explore other complex regulatory mechanisms at the exon level. Alternative splicing and polyadenylation play a crucial role in the functional diversity of a gene by generating different isoforms to regulate gene expression at the post-transcriptional level, and limiting analyses to the whole gene level can miss this important regulatory layer. Here, we demonstrate detailed step by step analyses for identification and visualization of differential exon and polyadenylation site usage across conditions, using Bioconductor and other packages and functions, including DEXSeq, diffSplice from the Limma package, and rMATS.

Alternative splicing (AS) and alternative polyadenylation (APA) expand the diversity of transcript isoforms and their products. Here, we describe bioinformatic protocols to analyze bulk RNA-seq and 3' end sequencing assays to detect and visualize AS and APA varying across experimental conditions.

RNA-seq数据中替代剪接和聚腺苷酸化的鉴定

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also ...

In Situ Hybridization Using Oligonucleotide Probes to Study RNA Expression in Rat Brain Sections

Source: Shokry, I. M., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/53165">Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome.</a>&#160;J. Vis. Exp.&#160;(2015).
This video demonstrates the detection of serotonin receptor RNA synthesis using oligonucleotide probes in rat brain tissue affected by serotonin syndrome. The protocol includes tissue pretreatment, probe hybridization, and enzymatic detection to visualize and quantify serotonin receptor RNA expression under a microscope.

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

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