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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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。
RNA分离步骤需要额外考虑。我们建议使用用 RNase 去除溶液预处理的层流式洁净空间。除了RNA产量外，还必须检查RNA的完整性，以确保样品在解剖和分离步骤中没有降解。RNA的完整性可以通过变性琼脂糖凝胶电泳来观察。果 蝇 的总 RNA 表现出单一的条带谱，因为 28S rRNA 解离成两个大小相等的亚基，它们与 18S rRNA25 结合。当使用单链RNA分子量标准时，这可以看作是大约600个核苷酸的单条带（图2A）。
分离的RNA样品进行G / I拖尾（图2B）。通过酵母聚（A）聚合酶（PAP）17在RNA的3'-末端添加鸟苷和肌苷残基，从而保护RNA的3'-末端免受酶降解。新合成的受 G/I 标签保护的全长 RNA 使用通用反向引物（3' 标记的 C10T2）进行逆转录以产生 cDNA 样品。然后使用多聚腺苷酸化位点上游的基因特异性正向/反向引物进行PCR，以产生显示尖锐条带的PCR产物。来自基因特异性正向引物和通用反向引物的PCR反应在凝胶上产生未定义长度的DNA分子，这反映了不同长度的poly（A）尾部。随后通过毛细管凝胶电泳分析PCR产物。
在设计引物时，需要注意的是，基因特异性正向/反向引物的扩增子大小应在 50-300 bp 的范围内。测试多个正向引物可以有机会找到适用于poly（A）尾部评估的最佳引物。我们建议在预测的poly（A）起始位点上游为基因特异性产物设计反向引物，因为这为直接计算poly（A）尾部长度提供了机会。在进行PCR反应时，重要的是进行"无逆转录酶对照"，以排除来自受污染基因组DNA的扩增。强烈建议使用退火温度梯度进行PCR优化。鉴于这种方法的性质，分析可变多聚腺苷酸化 mRNA 物种的 poly（A） 尾长需要额外考虑。例如，许多基因经历选择性多聚腺苷酸化以产生不同的 3'UTR 物种26。我们建议使用不同的PCR引物组来研究单个3'UTR物种。
我们强烈建议在每次运行中将基因特异性PCR产物纳入毛细管凝胶电泳中，因为每次运行中存在一些核苷酸的潜在迁移差异。在计算poly（A）尾长时，必须使用基因特异性PCR的实际碱基对大小，而不是理论大小。
一些 RNA 经历额外的转录后修饰，包括末端尿苷化27。当 mRNA 尿苷化时，G/I 拖尾法可能不适合测量 poly（A） 尾长。这是因为通用反向引物可能无法有效地扩增在其 3'-末端具有非腺嘌呤核苷酸的 mRNA 物种。G/I 拖尾是一种基于 PCR 的方法，它通过更有效地扩增较短的片段可能会产生不必要的偏倚。因此，用户在解释结果时应谨慎。
使用这里介绍的方法，我们证明了 G/I 拖尾可以有效地用于测量 果蝇 神经系统和 S2 细胞的 poly（A） 尾巴长度。当与毛细管电泳结合使用时，该方法具有高度定量性。虽然高通量分析提供了全局poly（A）尾部分析，但基于低通量PCR的G/I拖尾方法对于单转录本分析、中试实验和其他方法的验证仍然非常有用。总之，方法的选择取决于具体的研究问题和目标。研究人员应选择最适合其实验需求和资源的方法。

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。

<table><tbody><tr><td>3-(N-morpholino) propanesulfonic acid (MOPS)</td><td>Research Product Internation (RPI)</td><td>M92020</td><td></td></tr><tr><td>Agilent High Sensitivity DNA Kit</td><td>Agilent Technologies</td><td>&amp;nbsp;5067-4626</td><td></td></tr><tr><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></tr><tr><td>Apex 100 bp-Low DNA Ladder</td><td>Genesee Scientific</td><td>19-109</td><td></td></tr><tr><td>Bioanalyzer</td><td>Agilent 2100 Bioanalyzer G2938C</td><td></td><td></td></tr><tr><td>Diethyl pyrocarbonate (DEPC)</td><td>Research Product Internation (RPI)&amp;nbsp;</td><td>D43060</td><td></td></tr><tr><td>DNA dye (Gel Loading Dye, Purple (6x)</td><td>New England biolabs&amp;nbsp;</td><td>B7024S</td><td></td></tr><tr><td>&lt;em&gt;Drosophila &lt;/em&gt;S2 cell line</td><td>Drosophila Genomics Resource Center stock #181</td><td></td><td></td></tr><tr><td>Drosophila Schneider&amp;rsquo;s Medium</td><td>Thermo Fisher Scientific</td><td>21720024</td><td></td></tr><tr><td>Ehidium bromide</td><td>Genesee scientific&amp;nbsp;</td><td>20-276</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Sigma-Aldrich</td><td>F4135</td><td></td></tr><tr><td>Forceps Dumont 5&amp;nbsp;&amp;nbsp;</td><td>Fine Science tools&amp;nbsp;</td><td>11254-20</td><td></td></tr><tr><td>Nuclease free water</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>AM9932</td><td></td></tr><tr><td>PBS 10x</td><td>Research Product Internation (RPI)&amp;nbsp;</td><td>P32200</td><td></td></tr><tr><td>Poly(A) Tail-Length Assay Kit</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>764551KT</td><td></td></tr><tr><td>RiboRuler Low Range RNA Ladder</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>SM1833</td><td></td></tr><tr><td>RNA Gel Loading Dye (2x)</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>R0641</td><td></td></tr><tr><td>RNA microprep kit</td><td>Zymoresearch&amp;nbsp;</td><td>R1050&amp;nbsp;</td><td></td></tr><tr><td>RNA miniprep kit</td><td>Zymoresearch&amp;nbsp;</td><td>R1055</td><td></td></tr><tr><td>Scissors-Vannas Spring Scissors - 2.5 mm Cutting Edge</td><td>Fine Science tools&amp;nbsp;</td><td>15000-08</td><td></td></tr><tr><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></tr></tbody></table>

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GAPDH和Dscam1基因特异性引物对的PCR产物显示出明显的单条带，而来自基因特异性/通用引物对的PCR产物显示出不同的涂片模式（图4A和表1），这表明GAPDH和Dscam1 mRNA的poly（A）长度不同。在图4B中，涂片分析用于获得GAPDH和Dscam1的平均poly（A）尾长。输出并修改电泳图以表示poly（A）尾部长度（图4C）。
同样，我们对 S2 细胞进行了 poly（A） 尾长分析（图 5）。为了确定 Dscam1 3'UTR 在缩短 poly（A） 尾巴中的作用，用含有 SV40 3'UTR 或 Dscam1 3'UTR 的 Dscam1 编码区的 DNA 质粒转染 S2 细胞。由于SV40 3'UTR已被广泛用作重组DNA质粒的最小转录终止和多聚腺苷酸化信号，因此选择最小SV40 3'UTR来控制Dscam1 3'UTR。提取总RNA，并进行RT-PCR、G/I拖尾和高分辨率毛细管电泳（图5）。结果表明，SV40 3'UTR质粒的poly（A）尾长分布与内源性GAPDH相似，而Dscam1 3'UTR质粒的poly（A）尾长分布与幼虫脑内源性Dscam1 mRNA相似。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66116/66116fig04.jpg"> 图 4:来自幼虫大脑的 GAPDH 和 Dscam1 的 Poly（A） 尾长测量。 （A） 毛细管凝胶电泳将基因特异性 PCR 产物分离为 GAPDH2 和 Dscam1 的离散条带（第一和第二泳道）。使用通用反向引物的PCR产物显示涂片分布（第三和第四泳道）。* 非特异性波段。（B） 使用拖尾分析计算平均多（A）尾长度。（C）从导出的csv文件中重建poly（A）尾长的分布。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66116/66116fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66116/66116fig05.jpg"> 图 5:转染 Dscam1 DNA 质粒的 Poly（A） 尾长测量。 培养的 果蝇 S2 细胞与含有 Dscam1 编码区和 Dscam1 或 SV40 的 3′UTR 的 Dscam1 构建体共转染。（A） 将基因特异性 PCR 产物的毛细管凝胶电泳图像显示为 SV40 （Dscam1-SV40 3'UTR） 和 Dscam1 （Dscam1-Dscam1 3'UTR） 的离散条带（第一和第二泳道）。使用通用反向引物的PCR产物显示涂片模式（第三和第四泳道）。* 非特异性波段。（B） 使用拖尾分析计算平均多（A）尾长度。（C）从导出的csv文件中重建poly（A）尾长分布。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66116/66116fig05large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>Dscam1</td>
		</tr><tr><td>前向底漆 （5'-3'） CGCAGCCACAACAATTGAATG</td>
		</tr><tr><td>反向底漆 （5'-3'） AAATAAAATCAAAATCATATATTTAGCAACTTATGAAC</td>
		</tr><tr><td>SV40型</td>
		</tr><tr><td>前向底漆 （5'-3'） CCACAAAGGAAAAAGCTGCAC</td>
		</tr><tr><td>反向引物 （5'-3'） TTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTG</td>
		</tr><tr><td>GAPDH公司</td>
		</tr><tr><td>前向底漆 （5'-3'） CACTTCAGAAACGGCCTGAAAATGGC</td>
		</tr><tr><td>反向引物 （5'-3'） AATATTTAAATGCTTATGAGTCGGCATTTTTTTAAAACTAC</td>
		</tr><tr><td>通用反向引物</td>
		</tr><tr><td>反向引物 （5'-3'） GGTAATACGACTCACTATAGCGAGACCCCCCCCCCTT</td>
		</tr></tbody></table>表1:该协议中使用的引物。 
补充文件1:溶液的组成和PCR的设置。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66116/66116_JOVE_Supp%20FIle%201.zip" target="_blank">请点击此处下载此文件。</a>

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

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

835 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

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

激光捕获显微切割技术的果蝇外周神经元

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

科研

神经科学

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 expression is rarely tissue-specific enough to allow for precise correlation of the circuit with behavioral readouts. To overcome this major hurdle, we recently developed the FINGR method to achieve a more restrictive Gal4 expression in the tissue of interest. The FINGR method has three components: 1) the traditional Gal4/UAS system; 2) a set of FLP/FRT-mediated Gal80 converting tools; and 3) enhancer-trap FLP (ET-FLP). Gal4 is used to define the primary neural circuitry of interest. Paring the Gal4 with a UAS-effector, such as UAS-MJD78Q or UAS-Shits, regulates the neuronal activity, which is in turn manifested by alterations in the fly behavior. With an additional UAS-reporter such as UAS-GFP, the neural circuit involved in the specific behavior can be simultaneously mapped for morphological analysis. For Gal4 lines with broad expression, Gal4 expression can be restricted by using two complementary Gal80-converting tools: tubP&gt;Gal80&gt; ('flip out') and tubP&gt;stop&gt;Gal80 ('flip in'). Finally, investigators can turn Gal80 on or off, respectively, with the help of tissue-specific ET-FLP. In the flip-in mode, Gal80 will repress Gal4 expression wherever Gal4 and ET-FLP intersect. In the flip-out mode, Gal80 will relieve Gal4 repression in cells in which Gal4 and FLP overlap. Both approaches enable the restriction of the number of cells in the Gal4-defined circuitry, but in an inverse pattern. The FINGR method is compatible with the vast collection of Gal4 lines in the fly community and highly versatile for traditional clonal analysis and for neural circuit mapping. In this protocol, we demonstrate the mapping of FLP expression patterns in select ET-FLPx2 lines and the effectiveness of the FINGR method in photoreceptor cells. The principle of the FINGR method should also be applicable to other genetic model organisms in which Gal4/UAS, Gal80, and FLP/FRT are used.

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

We describe a Flippase-induced intersectional Gal80/Gal4 repression (FINGR) method, allowing tissue-specific FLP to determine Gal80 expression patterns. Wherever Gal4 and FLP overlap, Gal4 expression is turned on (Gal80 flipped out) or off (Gal80 flipped in). The FINGR method is versatile for clonal analysis and neural circuit mapping.

幼虫和成人测绘和增强子陷阱Flippase表达中的应用果蝇中枢神经系统

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 ...

来自 黑腹果蝇 第三龄幼虫的活脑外植体成像

Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a differentiating ganglion mother cell (GMC), which will undergo one additional division to give rise to two neurons or glia. Studies in NBs have uncovered the molecular mechanisms underlying cell polarity, spindle orientation, neural stem cell self-renewal, and differentiation. These asymmetric cell divisions are readily observable via live-cell imaging, making larval NBs ideally suited for investigating the spatiotemporal dynamics of asymmetric cell division in living tissue. When properly dissected and imaged in nutrient-supplemented medium, NBs in explant brains robustly divide for 12-20 h. Previously described methods are technically difficult and may be challenging to those new to the field. Here, a protocol is described for the preparation, dissection, mounting, and imaging of live third-instar larval brain explants using fat body supplements. Potential problems are also discussed, and examples are provided for how this technique can be used.

作者聚焦：研究不对称细胞分裂动力学：果蝇幼虫脑外植体实时成像的方案 

Here, we discuss a workflow to prepare, dissect, mount, and image live explant brains from Drosophila melanogaster third instar larvae to observe the cellular and subcellular dynamics under physiological conditions.

黑腹果蝇第三龄幼虫脑的活细胞成像

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a ...

发育生物学

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and amyotrophic lateral sclerosis (ALS). Protein aggregates are closely associated with neuropathology in these diseases, although the exact mechanism by which aberrant protein aggregation disrupts normal cellular homeostasis is not known. Emerging data provide strong support for the hypothesis that pathogenic aggregates in AD, PD, HD, and ALS have many similarities to prions, which are protein-only infectious agents responsible for the transmissible spongiform encephalopathies. Prions self-replicate by templating the conversion of natively-folded versions of the same protein, causing spread of the aggregation phenotype. How prions and prion-like proteins in AD, PD, HD, and ALS move from one cell to another is currently an area of intense investigation. Here, a Drosophila melanogaster model that permits monitoring of prion-like, cell-to-cell transmission of mutant huntingtin (Htt) aggregates associated with HD is described. This model takes advantage of powerful tools for manipulating transgene expression in many different Drosophila tissues and utilizes a fluorescently-tagged cytoplasmic protein to directly report prion-like transfer of mutant Htt aggregates. Importantly, the approach we describe here can be used to identify novel genes and pathways that mediate spreading of protein aggregates between diverse cell types in vivo. Information gained from these studies will expand the limited understanding of the pathogenic mechanisms that underlie neurodegenerative diseases and reveal new opportunities for therapeutic intervention.

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Accumulating evidence supports the idea that pathogenic protein aggregates associated with neurodegenerative diseases spread between cells with prion-like properties. Here, we describe a method that enables visualization of cell-to-cell spreading of prion-like aggregates in the model organism, Drosophila melanogaster.

监测果蝇中朊病毒样蛋白聚集物的细胞间传输

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

嗜德维人神经元树突状树突状复杂度的定量分析

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

荧光标记蛋白 ImageJ 的无偏分析在果蝇变性定量细胞生物学中的应用

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

成绩单和代谢产物的分离纯化<em&gt;果蝇</em&gt;头

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

生物学

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. Furthermore, despite progress in understanding postsynaptic MTs, much less is known about the contributions of presynaptic MTs to neuronal morphogenesis. In particular, studies of in vivo MT dynamics in Drosophila sensory dendrites yielded significant insights into polymer-level behavior. However, the technical and analytical challenges associated with live imaging of the fly neuromuscular junction (NMJ) have limited comparable studies of presynaptic MT dynamics. Moreover, while there are many highly effective software strategies for automated analysis of MT dynamics in vitro and ex vivo, in vivo data often necessitate significant operator input or entirely manual analysis due to inherently inferior signal-to-noise ratio in images and complex cellular morphology. &#160;To address this, this study optimized a new software platform for automated and unbiased in vivo particle detection. Multiparametric analysis of live time-lapse confocal images of EB1-GFP labeled MTs was performed in both dendrites and the NMJ of Drosophila larvae and found striking differences in MT behaviors. MT dynamics were furthermore analyzed following knockdown of the MT-associated protein (MAP) dTACC, a key regulator of Drosophila synapse development, and identified statistically significant changes in MT dynamics compared to wild type. These results demonstrate that this novel strategy for the automated multiparametric analysis of both pre- and postsynaptic MT dynamics at the polymer-level significantly reduces human-in-the-loop criteria. The study furthermore shows the utility of this method in detecting distinct MT behaviors upon dTACC-knockdown, indicating a possible future application for functional screens of factors that regulate MT dynamics in vivo. Future applications of this method may also focus on elucidating cell type and/or compartment-specific MT behaviors, and multicolor correlative imaging of EB1-GFP with other cellular and subcellular markers of interest.&#160;

This study presents a noninvasive intravital neuronal imaging strategy combined with a new software strategy to achieve automated, unbiased tracking and analysis of in vivo microtubule (MT) plus-end dynamics in the sensory dendrites and the neuromuscular junctions of Drosophila.

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. ...

果蝇幼虫脑和细胞系的Poly A尾长测量

副本

摘要

探索更多视频

此视频中的章节

激光捕获显微切割技术的果蝇外周神经元

幼虫和成人测绘和增强子陷阱Flippase表达中的应用果蝇中枢神经系统

黑腹果蝇第三龄幼虫脑的活细胞成像

黑腹果蝇第三龄幼虫脑的活细胞成像

黑腹果蝇第三龄幼虫脑的活细胞成像

监测果蝇中朊病毒样蛋白聚集物的细胞间传输

嗜德维人神经元树突状树突状复杂度的定量分析

荧光标记蛋白 ImageJ 的无偏分析在果蝇变性定量细胞生物学中的应用

成绩单和代谢产物的分离纯化

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

果 蝇 幼虫脑和细胞系的Poly A尾长测量

副本

摘要

探索更多视频

此视频中的章节

激光捕获显微切割技术的果蝇外周神经元

幼虫和成人测绘和增强子陷阱Flippase表达中的应用果蝇中枢神经系统

黑腹果蝇第三龄幼虫脑的活细胞成像

黑腹果蝇第三龄幼虫脑的活细胞成像

黑腹果蝇第三龄幼虫脑的活细胞成像

监测果蝇中朊病毒样蛋白聚集物的细胞间传输

嗜德维人神经元树突状树突状复杂度的定量分析

荧光标记蛋白 ImageJ 的无偏分析在果蝇变性定量细胞生物学中的应用

成绩单和代谢产物的分离纯化

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

果蝇幼虫脑和细胞系的Poly A尾长测量