这项研究得到了美国国家神经疾病和中风研究所R01NS116463 JK 的支持，以及内华达大学里诺分校的细胞和分子成像核心设施，该设施得到了美国国立卫生研究院资助P20GM103650的支持，并用于本研究中报告的研究。

1. 饲养和选择果蝇幼虫
<ol><li>在加湿培养箱中，在25°C的标准苍蝇食品培养基上维持/培养苍蝇菌株（w1118，野生型）。</li>
	<li>选10只游荡的3龄幼虫产卵后72小时。</li>
	<li>将幼虫放入35毫米的空培养皿中，并使用镊子将幼虫转移到装有自来水的新培养皿中，轻轻清洗它们。执行此操作 2 次以去除任何剩余的食物。</li>
</ol>2.从果蝇幼虫的大脑分离（图1）
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66116/66116fig01.jpg"> 图1:3龄游荡期果蝇幼虫脑的解剖。 （A）果蝇幼虫示意图。（B-G）幼虫解剖。<a href="https://www.jove.com/files/ftp_upload/66116/66116fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<ol><li>将10只幼虫放在含有冰冷PBS的解剖盘上。</li>
	<li>将幼虫背侧朝上放置（通过沿其长度延伸的气管插管识别），并将每一端固定在培养皿的底部，然后在后端的体壁上做一个小切口。</li>
	<li>使用显微解剖剪刀沿背中线向前端切割体壁。</li>
	<li>使用巴斯德移液管在培养皿中用PBS进行3次短暂冲洗幼虫内部，以暴露大脑。</li>
	<li>使用镊子定位并抬起大脑，并使用显微解剖剪刀小心地将其隔离。</li>
	<li>将解剖的大脑转移到1.5mL微量离心管中，该离心管在冰上装满了冰冷的PBS;收集所有幼虫的大脑。如第 4 节所述，使用 RNA 微制备试剂盒进行 RNA 提取。 注意:在15分钟内解剖10只幼虫，以防止组织损伤和RNA降解。</li>
</ol>3. 果蝇 S2 施耐德细胞
<ol><li>在25°C下，在25°C下在补充有10%胎牛血清（FBS）的果蝇Schneider培养基中培养果蝇S2细胞，密度为8×106至10 ×10 6个细胞/ mL，活力至少为90%。</li>
	<li>在 50 mL 无菌锥形管中，用补充有 10% FBS 的施耐德果蝇培养基将细胞稀释至 2.5 ×10 6 个细胞/mL，该培养基已预热至 25 °C。</li>
	<li>将 8 mL 细胞悬液（20 × 106 个细胞）转移到 100 mm 培养板中，并加入 4 mL 培养基使其达到 12 mL（第 1 天）。</li>
	<li>将培养的细胞在25°C下在湿润培养箱中孵育。 注意:细胞在12-16小时（第2天）后松散地粘附在板上。</li>
	<li>用适当的 DNA 质粒转染细胞24.</li>
	<li>在加湿培养箱中孵育48小时。</li>
	<li>孵育后，通过轻轻移液（第 4 天）加入 5 mL 冰冷的 PBS 来收集细胞。</li>
	<li>将细胞转移到 15 mL 试管中。</li>
	<li>通过在4°C下以1,000× g 离心5分钟来沉淀细胞。</li>
	<li>通过轻轻移液用冰冷的PBS冲洗细胞2次，并通过在4°C下以1,000× g 离心5分钟来收集细胞。</li>
	<li>使用 RNA 小量制备试剂盒进行 RNA 提取。 注意: 在无菌层流罩内执行以下步骤。</li>
</ol>4. 从果蝇幼虫脑和 S2 细胞中提取总 RNA
<ol><li>幼虫脑: 通过短暂离心（在5,000× g下短旋8秒）除去PBS。</li>
	<li>加入 600 μL RNA 裂解缓冲液，并用塑料研杵匀浆 10 倍。在体视显微镜下目视检查试管以确保完全裂解。</li>
	<li>在4°C下以1,000× g 离心5分钟以除去组织碎片。将清除的上清液转移到无核酸酶的微量离心管中。</li>
	<li>根据制造商的说明，使用 RNA 微量制备试剂盒分离 RNA。 注意:由于样品中存在少量 RNA，因此使用 RNA 微制备试剂盒对于幼虫脑样品至关重要。</li>
	<li>S2 细胞: 根据制造商的说明去除 PBS 并分离 RNA。</li>
	<li>通过分光光度法和琼脂糖凝胶电泳测量RNA产量和质量。<ol><li>通过分别在A260nm 和A280nm处测量提取的RNA的光密度来确定提取的RNA的纯度和数量。确保 A260 nm/A 280 nm 比值为 ≥2.0，下游应用的 RNA 浓度为 >350 ng/μL。 注:10 个 果蝇 幼虫大脑的典型 RNA 产量为 ~500-800 ng/μL 或 5 μL 中的 2.5-4 μg。对于 S2 细胞，产率为 ~2-3 μg/μL（15 μL 中为 15-30 μg）。分离的RNA可以储存在-80°C下进行长期储存。</li>
	</ol></li>
</ol>5. RNA凝胶的制备和电泳
<ol><li>1.5% 变性 RNA 凝胶 （100 mL） 注意:甲醛通过皮肤接触和吸入蒸气有毒;在化学通风橱中处理它。<ol><li>将三片琼脂糖片剂（1.5g）溶解在82mL MOPS缓冲液（补充文件1）中，直至片剂完全分解形成细颗粒。</li>
		<li>在微波炉中加热琼脂糖浆料，直到溶液澄清并且所有颗粒完全溶解。</li>
		<li>将溶液冷却至~60°C。</li>
		<li>加入 18 mL 37% 甲醛，然后轻轻旋转混合。将溶液倒入铸造托盘中，使其在通风橱中凝固。</li>
	</ol></li>
	<li>RNA样品制备和电泳<ol><li>将 RNA 样品稀释至 200 ng（5 μL 中），并加入 5 μL 2x RNA 上样染料。</li>
		<li>在干浴中将样品在70°C下加热5分钟。</li>
		<li>在第一泳道中加载 2 μL RNA 分子量标准，在相邻泳道中加载 10 μL 样品。</li>
		<li>在100V的MOPS缓冲液中进行电泳60分钟，以获得5×6cm凝胶。 注意:根据扩增子大小调整电泳条件。</li>
		<li>在紫外透射仪上可视化凝胶。 注意:单条带~600核苷酸大小的存在表明RNA制备完整（见 图2A）。</li>
	</ol></li>
</ol>6. Poly（A）尾长测量
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66116/66116fig02v2.jpg"> 图 2:RNA 样品制备和 poly（A）-tail 测定。 （A） RNA 凝胶图像显示 1.5% 甲醛琼脂糖凝胶上来自 果蝇 幼虫脑（左）和 S2 细胞（右）的总 RNA。单链RNA分子量标准大小显示在泳道M上的核苷酸中，注意~600 nt处的主要RNA条带，它来自rRNA。（B） poly（A）-tail测定示意图。 缩写:G/I = 鸟苷/肌苷。 <a href="https://www.jove.com/files/ftp_upload/66116/66116fig02largev2.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<ol><li>GI拖尾（图2B）<ol><li>将试剂保持在冰上，制备以下混合物 （20 μL）:最多 14 μL 总 RNA 样品 （1 μg）、4 μL 5x Tail 缓冲液混合物和 2 μL 10x Tail 酶混合物。</li>
		<li>在37°C下在热循环仪中孵育60分钟。</li>
		<li>加入 1.5 μL 尾部止动液;在冰上保持2分钟。 注意:继续逆转录或将GI尾RNA样品储存在-80°C，直到准备好进行逆转录。</li>
	</ol></li>
	<li>逆转录和PCR扩增<ol><li>通过制备混合物并在 补充文件 1 中描述的条件下孵育来合成 cDNA。</li>
		<li>稀释 cDNA 样品并进行 PCR 以扩增 DNA，如 补充文件 1 所示。</li>
	</ol></li>
</ol>7. 琼脂糖凝胶电泳法PCR产物分析
<ol><li>通过在100V下电泳45分钟，在2.5%琼脂糖凝胶上分析步骤6.2.2中的一小部分（2-5μL）PCR产物，以进行质量控制。</li>
	<li>通过对凝胶提取的PCR条带进行测序，验证PCR对基因特异性和尾部特异性反应的特异性。</li>
</ol>8. 毛细管电泳
<ol><li>使用带有高灵敏度 DNA 试剂盒的生物分析仪对来自基因特异性和多 （A） 特异性 PCR 的 1 μL PCR 产物 （0.5-5 ng/μL） 进行高分辨率凝胶电泳。寻找分辨率高的峰值，表明运行成功。</li>
</ol>9. 数据分析:poly（A）尾长测量（图3）
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66116/66116fig03v2.jpg"> 图 3:Poly（A） 尾部长度和峰值测量。 <a href="https://www.jove.com/files/ftp_upload/66116/66116fig03largev2.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<ol><li>访问数据<ol><li>要访问数据，请在软件中打开 xad 文件。</li>
		<li>在树视图面板中选择示例名称或梯形图。 注意:这将以所选样品的电泳图或凝胶状图像的形式显示结果。下标记 35 碱基对 （bp） 和上标记 10,380 bp 是用于将标准数据 （50-7,000 bp） 与样品孔数据对齐的内标。</li>
		<li>放大和缩小电泳图和凝胶状图像以显示细节。</li>
	</ol></li>
	<li>执行峰和涂片分析<ol><li>要获得峰尺寸，请打开所选样品的电泳图。</li>
		<li>右键单击电泳图并选择 手动积分 ，通过拖动水平线手动选择峰。</li>
		<li>观察 "峰值"表中的峰值。识别具有最大峰高的峰。这是单个样品的poly（A）尾长峰。在所示示例中，它是 346 bp。</li>
		<li>启用顶部菜单上的 "显示/隐藏设定点 "图标，然后等待右侧弹出一个新面板。</li>
		<li>选择 "高级"，向下滚动以找到 "执行拖尾分析"，然后选中该复选框。这会将 Region 表 添加到电泳图选项卡中。</li>
		<li>从样品中选择一个电泳图，然后转到 "区域"（Region） 表，该表显示 "从 [bp] "和 "到"[bp]" 菜单。要设置开始和结束 [bp]，请右键单击 电泳图 ，然后选择 "区域 "以添加 "添加区域"。</li>
		<li>右键单击 "区域"（Region） 表格 中的任何单元格，然后选择 "修改区域"（Modify Regions ），弹出一个小的新窗口，可在其中设置自定义区域。 注:例如，我们对 GAPDH 使用了 300 bp 至 550 bp 的区域。基因特异性GAPDH PCR在265 bp处产生峰值。通用引物（表1）通过退火到G/I-tailed RNA将poly（A）PCR的长度延长35 bp。因此，GAPDH RNA 上的第一个腺嘌呤核苷酸从 300 bp （265 + 35） 开始。我们任意将最大poly（A）尾部长度限制为250（300 + 250 = 550）。从区域表中，程序返回区域内的平均大小为 387 bp。</li>
		<li>使用公式 （1） 计算目标 mRNA 上的 poly（A） 尾长: Poly（A） 尾长 = （A - B - 35） （1） 其中 A 是电泳图中 poly（A） 特异性 PCR 产物的平均 bp（即 GAPDH 为 387 bp），B 是电泳图中基因特异性 PCR 产物的峰值 bp（即 GAPDH 为 265 bp），"35"是通用反向引物标签的长度。 注:根据上述计算，GAPDH 的平均 poly（A） 尾长为 387 - 265 - 35= 87 bp。</li>
	</ol></li>
</ol>10. 可视化poly（A）尾长分布
<ol><li>将数据导出为 csv 文件，位于 "文件"|"导出 |用于检索示例数据的示例数据 。 注意:导出的 csv 文件在 X 轴上显示 运行时间 而不是 bp 。</li>
	<li>转到样品的电泳图，然后选中"电泳图"选项卡上的"显示尺寸"，以自动将"区域表"上的区域表从 bp 转换为运行时。在本例中，70.39 秒至 86.28 秒对应于 300 bp 至 550 bp。</li>
	<li>打开 csv 文件，选择运行时间 的 70.39 秒 到 86.28 秒 之间的值以生成图形。要在图表上将 bp 大小可视化到 X 轴，请将具有 bp 大小的电泳图导出为图像文件，并将其叠加在电子表格中生成的图表上。这将适当匹配 poly（A） 尾部分布的 bp 大小。</li>
</ol>

量化 果蝇 神经系统基因的 Poly（A） 长度

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+G-tailing+method+for+the+determination+of+the+poly(a)+tail+length+applied+to+hepatitis+a+virus+RNA.">A new G-tailing method for the determination of the poly(a) tail length applied to hepatitis a virus RNA.</a> Nucleic Acids Research. 29 (12), 57 (2001).</li><li>Bazzini, A. A., Lee, M. T., Giraldez, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+profiling+shows+that+miR-430+reduces+translation+before+causing+mRNA+decay+in+zebrafish.">Ribosome profiling shows that miR-430 reduces translation before causing mRNA decay in zebrafish.</a> Science. 336 (6078), 233-237 (2012).</li><li>Subtelny, A. O., Eichhorn, S. W., Chen, G. R., Sive, H., Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(a)-tail+profiling+reveals+an+embryonic+switch+in+translational+control.">Poly(a)-tail profiling reveals an embryonic switch in translational control.</a> Nature. 508 (1), 66-71 (2014).</li><li>Chang, H., Lim, J., Ha, M., Kim, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TAIL-seq:+Genome-wide+determination+of+poly(a)+tail+length+and+3'+end+modifications.">TAIL-seq: Genome-wide determination of poly(a) tail length and 3' end modifications.</a> Molecular Cell. 53 (6), 1044-1052 (2014).</li><li>Legnini, I., Alles, J., Karaiskos, N., Ayoub, S., Rajewsky, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FLAM-seq:+Full-length+mRNA+sequencing+reveals+principles+of+poly(A)+tail+length+control.">FLAM-seq: Full-length mRNA sequencing reveals principles of poly(A) tail length control.</a> Nature Methods. 16 (9), 879-886 (2019).</li><li>Garalde, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+parallel+direct+RNA+sequencing+on+an+array+of+nanopores.">Highly parallel direct RNA sequencing on an array of nanopores.</a> Nature Methods. 15 (3), 201-206 (2018).</li><li>Singh, M., Ye, B., Kim, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+leucine+zipper+kinase+regulates+Dscam+expression+through+a+noncanonical+function+of+the+cytoplasmic+poly(A)-binding+protein.">Dual leucine zipper kinase regulates Dscam expression through a noncanonical function of the cytoplasmic poly(A)-binding protein.</a> Journal of Neuroscience. 42 (31), 6007-6019 (2022).</li><li>Macharia, R. W., Ombura, F. L., Aroko, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insects'+RNA+profiling+reveals+absence+of+&amp;#34;hidden+break&amp;#34;+in+28S+ribosomal+RNA+molecule+of+onion+thrips,+Thrips+tabaci.">Insects' RNA profiling reveals absence of &amp;#34;hidden break&amp;#34; in 28S ribosomal RNA molecule of onion thrips, Thrips tabaci.</a> Journal of Nucleic Acids. 2015, 965294 (2015).</li><li>Miura, P., Sanfilippo, P., Shenker, S., Lai, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+polyadenylation+in+the+nervous+system:+to+what+lengths+will+3'+UTR+extensions+take+us.">Alternative polyadenylation in the nervous system: to what lengths will 3' UTR extensions take us.</a> Bioessays. 36 (8), 766-777 (2014).</li><li>Sement, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al+Uridylation+prevents+3'+trimming+of+oligoadenylated+mRNAs.">et al Uridylation prevents 3' trimming of oligoadenylated mRNAs.</a> Nucleic Acids Research. 41 (14), 7115-7127 (2013).</li></ol>

作者没有需要披露的利益冲突。

在该方案中，我们描述了从徘徊的3龄阶段解剖果蝇幼虫大脑的技术以及果蝇S2细胞的样品制备。由于 mRNA 的不稳定性，样本采集需要格外小心。对于幼虫脑夹层，大脑在隔离期间不应受损，也不应长时间保存在溶液中。将解剖时间保持在 8-10 分钟进行一轮解剖是必不可少的。用RNA酶抑制剂补充解剖液也可能是有益的。至于 S2 细胞培养和转染，请在层流罩中执行所有步骤。...

大多数真核 mRNA 通过经典 poly（A） 聚合酶添加非模板化腺苷，在细胞核的 3' 末端进行转录后多聚腺苷化。完整的 poly（A） 尾在 mRNA 的整个生命周期中至关重要，因为它对 mRNA 核输出至关重要1，促进与 poly（A） 结合蛋白的相互作用以提高翻译效率2，并赋予抗降解性3。在某些情况下，poly（A） 尾部也可以在细胞质中延伸，这是由非经典 poly（A） 聚合酶4 促进的。在细胞质中，poly （A） 尾长动态变化并影响 mRNA 分子的寿命。已知许多聚合酶和脱烯酰化酶可调节尾长 5,6,7。例如，poly（A）尾巴的缩短与翻译抑制相关，而poly（A）尾巴的延长增强了翻译8,9。
不断积累的基因组研究已经证明了poly（A）尾长在真核生物学各个方面的基本意义。这包括在生殖细胞发育、早期胚胎发育、学习和记忆的神经元突触可塑性以及炎症反应中的作用10。已经开发了许多用于测量poly（A）尾部长度的方法和测定方法。例如，RNase H/oligo（dT）测定利用RNase H在存在或不存在oligo（dT）的情况下研究poly（A）尾长11,12。研究poly（A）尾部的其他方法包括3'末端的PCR扩增，例如cDNA末端poly（A）测试（RACE-PAT）的快速扩增（RACE-PAT）12,13和连接酶介导的poly（A）测试（LM-PAT）14。PAT测定的进一步修改包括ePAT15和sPAT16。酶促 G 尾17,18 或 3' 端的 G/I 尾是 PAT 测定的其他变体。这些技术的进一步修改包括使用荧光标记引物以及毛细管凝胶电泳进行高分辨率分析，称为高分辨率poly（A）测试（Hire-PAT）19。这些 PCR 驱动的检测可实现快速、高灵敏度的 poly（A） 长度定量。
随着下一代测序的发展，PAL-seq20 和 TAIL-seq21 等高通量测序方法允许在转录组范围内进行多聚腺苷酸化分析。然而，这些方法仅提供 36-51 个核苷酸的短测序读数。因此，FLAM-Seq22 是为全长 mRNA 的全局尾长分析而开发的，并提供长读长。纳米孔技术23 提供 PCR 非依赖性、直接 RNA 或直接 cDNA 测序，用于 poly（A） 尾长估计。然而，这些高通量方法并非没有局限性。它们需要大量的起始材料，价格昂贵且耗时。此外，使用高通量方法分析稀有转录本可能极具挑战性，而当需要分析少量转录本、用于试点实验和其他方法的验证时，基于低通量PCR的方法仍然具有优势。
我们最近证明 Dscam1 mRNA 在果蝇中含有短的 poly（A） 尾巴，这需要使用 G/I 拖尾方法24 将细胞质 poly（A） 结合蛋白与 Dscam1 3'UTR 进行非经典结合。在这里，我们提供了一种简化的程序，用于组织制备和定量来自果蝇神经系统和果蝇 S2 细胞的 poly（A） 长度的 mRNA。

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			<td>M92020</td>
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			<td>Agilent High Sensitivity DNA Kit</td>
			<td>Agilent Technologies</td>
			<td>&#160;5067-4626</td>
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			<td>Agilent software 2100 expert free download demo</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td>https://www.agilent.com/en/product/automated-electrophoresis/bioanalyzer-systems/bioanalyzer-software/2100-expert-software-228259</td>
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			<td>Apex 100 bp-Low DNA Ladder</td>
			<td>Genesee Scientific</td>
			<td>19-109</td>
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			<td>Bioanalyzer</td>
			<td>Agilent 2100 Bioanalyzer G2938C</td>
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			<td>Diethyl pyrocarbonate (DEPC)</td>
			<td>Research Product Internation (RPI)&#160;</td>
			<td>D43060</td>
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			<td>DNA dye (Gel Loading Dye, Purple (6x)</td>
			<td>New England biolabs&#160;</td>
			<td>B7024S</td>
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			<td>Drosophila S2 cell line</td>
			<td>Drosophila Genomics Resource Center stock #181</td>
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			<td></td>
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			<td>Drosophila Schneider&#8217;s Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>21720024</td>
			<td></td>
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			<td>Ehidium bromide</td>
			<td>Genesee scientific&#160;</td>
			<td>20-276</td>
			<td></td>
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			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F4135</td>
			<td></td>
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			<td>Forceps Dumont 5&#160;&#160;</td>
			<td>Fine Science tools&#160;</td>
			<td>11254-20</td>
			<td></td>
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			<td>Nuclease free water</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>AM9932</td>
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			<td>PBS 10x</td>
			<td>Research Product Internation (RPI)&#160;</td>
			<td>P32200</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(A) Tail-Length Assay Kit</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>764551KT</td>
			<td></td>
		</tr>
		<tr>
			<td>RiboRuler Low Range RNA Ladder</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>SM1833</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA Gel Loading Dye (2x)</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>R0641</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA microprep kit</td>
			<td>Zymoresearch&#160;</td>
			<td>R1050&#160;</td>
			<td></td>
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			<td>RNA miniprep kit</td>
			<td>Zymoresearch&#160;</td>
			<td>R1055</td>
			<td></td>
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			<td>Scissors-Vannas Spring Scissors - 2.5 mm Cutting Edge</td>
			<td>Fine Science tools&#160;</td>
			<td>15000-08</td>
			<td></td>
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			<td>TopVision Agarose Tablets</td>
			<td>Thermo Fisher Scientific</td>
			<td>R2802</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-Acetate-EDTA (TAE)</td>
			<td>Thermo Fisher Scientific</td>
			<td>B49</td>
			<td></td>
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measurement of poly a tail length from drosophila larva brain and cell line

聚腺苷酸化是一种重要的转录后修饰，可将 poly（A） 尾部添加到 mRNA 分子的 3' 末端。poly（A）尾巴的长度受细胞过程的严格调节。mRNA 多聚腺苷酸化的失调与异常基因表达和各种疾病有关，包括癌症、神经系统疾病和发育异常。因此，了解多聚腺苷酸化的动力学对于揭示 mRNA 加工和转录后基因调控的复杂性至关重要。 
本文介绍了一种测量从 果蝇 幼虫脑和 果蝇 施耐德 S2 细胞中分离的 RNA 样品中 poly（A） 尾长的方法。我们采用鸟苷/肌苷 （G/I） 拖尾方法，该方法涉及使用酵母聚 （A） 聚合酶在 mRNA 的 3' 末端酶促添加 G/I 残基。这种修饰可保护 RNA 的 3' 末端免受酶降解。然后使用通用反义引物对受保护的全长poly（A）尾部进行反转录。随后，使用靶向目标基因的基因特异性寡核苷酸以及用于逆转录的通用序列寡核苷酸进行PCR扩增。 
这会产生包含目的基因的poly（A）尾部的PCR产物。由于聚腺苷酸化不是一种均匀的修饰，并且会导致不同长度的尾部，因此PCR产物显示出一系列尺寸，导致琼脂糖凝胶上的涂抹图案。最后，对PCR产物进行高分辨率毛细管凝胶电泳，然后使用poly（A）PCR产物和基因特异性PCR产物的大小进行定量。该技术为分析poly（A）尾长提供了一种简单可靠的工具，使我们能够更深入地了解控制mRNA调控的复杂机制。

在这里，我们分析了果蝇幼虫大脑中 Dscam1 和 GAPDH 的 poly（A） 尾长（图 4）。分离的RNA在琼脂糖凝胶上可视化，用于质量控制。大约 600 个核苷酸大小的单个 RNA 条带表示完整的 RNA 制备（图 2A）。使用 Agilent 2100 生物分析仪对 RNA 进行 G/I 拖尾和高分辨率毛细管电泳。使用 Agilent 2100 Expert 软件导出凝胶图像并进行相应组装。来自GAP...

Watch this Scientific Journal Video about Measurement of Poly A Tail Length from Drosophila Larva Brain and Cell Line at JoVE.com

作者聚焦:使用 Poly A Tail 分析技术探索 mRNA 研究的前沿

该协议描述了一种有效且可靠的方法，用于量化 果蝇 神经系统中目的基因的poly（A）长度，该方法可以很容易地适应来自其他物种的组织或细胞类型。

果 蝇 幼虫脑和细胞系的Poly A尾长测量

Polyadenylation is a crucial posttranscriptional modification that adds poly(A) tails to the 3' end of mRNA molecules. The length of the poly(A) tail is ...

多聚腺苷酸化是一种转录后修饰。这将poly-A尾部添加到mRNA分子的三素端。多聚腺苷酸化的失调与基因表达异常和各种疾病有关，包括神经系统疾病。我们的方法为测量聚A长度提供了一种经济高效、高分辨率的分析。poly-A尾部分析的高通量方法（如二代测序）和高通量测序可提供高内涵信息。然而，使用这些方法分析稀有转录本可能极具挑战性。基于PCR的方法通常是低通量的。但是，它们可以很容易地用于分析少量转录本。GI拖尾法就是其中之一。通过这种方法，我们发现 Dscam mRNA 含有短的 poly-A 尾巴。Dscam 是一种神经元细胞粘附分子。它参与许多重要的神经发育过程。由于 Dscam mRNA 与大多数 mRNA 不同，含有短的 poly-A 尾巴，因此 Dscam mRNA 翻译处于独特的基因调控之下，这是我们试图回答的问题之一。

measurement-poly-tail-length-from-drosophila-larva-brain-cell

Research

JoVE Journal

Neuroscience

Measurement of Poly A Tail Length from Drosophila Larva Brain and Cell Line

819 次观看.  University of Nevada, Reno. 多聚腺苷酸化是一种转录后修饰。这将poly-A尾部添加到mRNA分子的三素端。多聚腺苷酸化的失调与基因表达异常和各种疾病有关，包括神经系统疾病。我们的方法为测量聚A长度提供了一种经济高效、高分辨率的分析。poly-A尾部分析的高通量方法（如二代测序）和高通量测序可提供高内涵信息。然而，使用这些方法分析稀有转录本可能极具挑战性。基于PCR的方法通常?...

视频: 作者聚焦:使用 Poly A Tail 分析技术探索 mRNA 研究的前沿

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval body wall contains several cell types that are models to study cell and tissue morphogenesis. For example tracheae are used to examine tube morphogenesis1, and the dendritic arborization (DA) sensory neurons of the Drosophila larva have become a primary system for the elucidation of general and neuron-class-specific mechanisms of dendritic differentiation2-5 and degeneration6.
The shape of dendrite branches can vary significantly between neuron classes, and even among different branches of a single neuron7,8. Genetic studies in DA neurons suggest that differential cytoskeletal organization can underlie morphological differences in dendritic branch shape4,9-11. We provide a robust immunological labeling method to assay in vivo microtubule organization in DA sensory neuron dendrite arbor (Figures 1, 2, Movie 1). This protocol illustrates the dissection and immunostaining of first instar larva, a stage when active sensory neuron dendrite outgrowth and branching organization is occurring 12,13.
In addition to staining sensory neurons, this method achieves robust labeling of microtubule organization in muscles (Movies 2, 3), trachea (Figure 3, Movie 3), and other body wall tissues. It is valuable for investigators wishing to analyze microtubule organization in situ in the body wall when investigating mechanisms that control tissue and cell shape.

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how complex cell shapes, such as neuronal dendrites, are achieved during development, it is important to be able to accurately assay microtubule organization. Here we describe a robust immunohistological labeling method to examine microtubule organization of dendritic arborization neuron sensory dendrites, trachea, muscle, and other Drosophila larva body wall tissues.

免疫组化标记在感觉神经元树突的微管，气管，并在肌肉果蝇幼虫体壁

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval ...

科研

神经科学

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

代的永生化小鼠脑微血管内皮细胞株作为<em在体外</em血脑屏障型号

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

方法对敌人造成闭合性颅脑创伤性脑损伤<em&gt;果蝇</em

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

觅食路径长度协议<em&gt;果蝇</em&gt;幼虫

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

全细胞电压钳记录的电生理方法<em&gt;果蝇</em&gt;光感受器

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

实时 cAMP 检测气味受体激活的活细胞测量

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

人类成人皮肤成纤维细胞诱导神经元高产培养的简单生成

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

在体内果蝇前运动神经末端的单分子跟踪

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these mechanisms is crucial for developing neural regenerative therapies. Drosophila melanogaster is a leading model for studies of neural development but historically has not been exploited to investigate adult brain regeneration. This is primarily because the adult brain exhibits very low mitotic activity. Nonetheless, penetrating traumatic brain injury (PTBI) to the adult Drosophila central brain triggers the generation of new neurons and new glia. The powerful genetic tools available in Drosophila combined with the simple but rigorous injury protocol described here now make adult Drosophila brain a robust model for neural regeneration research. Provided here are detailed instructions for (1) penetrating injuries to the adult central brain and (2) dissection, immunohistochemistry, and imaging post-injury. These protocols yield highly reproducible results and will facilitate additional studies to dissect mechanisms underlying neural regeneration.

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

This article provides detailed protocols for inflicting Penetrating Traumatic Brain Injury (PTBI) to adult Drosophila and examining the resulting neurogenesis.

刺激和分析 果蝇 中枢脑中的成人神经发生

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these ...

Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants

Neural stem cells (NSCs) have the ability to proliferate, differentiate, undergo apoptosis, and even enter and exit quiescence. Many of these processes are controlled by the complex interplay between NSC intrinsic genetic programs with NSC extrinsic factors, local and systemic. In the genetic model organism, Drosophila melanogaster, NSCs, known as neuroblasts (NBs), switch from quiescence to proliferation during the embryonic to larval transition. During this time, larvae emerge from their eggshells and begin crawling, seeking out dietary nutrients. In response to animal feeding, the fat body, an endocrine organ with lipid storage capacity, produces a signal, which is released systemically into the circulating hemolymph. In response to the fat body-derived signal (FBDS), Drosophila insulin-like peptides (Dilps) are produced and released from brain neurosecretory neurons and glia, leading to downstream activation of PI3-kinase growth signaling in NBs and their glial and tracheal niche. Although this is the current model for how NBs switch from quiescence to proliferation, the nature of the FBDS extrinsic cue remains elusive. To better understand how NB extrinsic systemic cues regulate exit from quiescence, a method was developed to culture early larval brains in vitro before animal feeding. With this method, exogenous factors can be supplied to the culture media and NB exit from quiescence assayed. We found that exogenous insulin is sufficient to reactivate NBs from quiescence in whole-brain explants. Because this method is well-suited for large-scale screens, we aim to identify additional extrinsic cues that regulate NB quiescence versus proliferation decisions. Because the genes and pathways that regulate NSC proliferation decisions are evolutionarily conserved, results from this assay could provide insight into improving regenerative therapies in the clinic.

Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants

A method to reactivate quiescent neural stem cells in cultured Drosophila brain explants has been established. Using this method, the role of systemic signals can be uncoupled from tissue-intrinsic signals in the regulation of neural stem cell quiescence, entry and exit.

培养的 果蝇 脑外植体中的神经干细胞再激活

Neural stem cells (NSCs) have the ability to proliferate, differentiate, undergo apoptosis, and even enter and exit quiescence. Many of these processes ...

果 蝇 幼虫脑和细胞系的Poly A尾长测量

果蝇幼虫脑和细胞系的Poly A尾长测量