Name: enhanced northern blot detection of small rna species in drosophila melanogaster
Uploaded: 2014-08-21T15:00:00.000+00:00
Duration: 9 min 39 s
Description: Watch this Scientific Journal Video about Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster at JoVE.com

这项工作是由国家研究所德拉桑特等德拉RECHERCHEMédicale的支持下，该中心的法国国家科学研究中，圣圣斯特拉斯堡总医院斯特拉斯堡，协会倒拉RECHERCHE河畔勒癌，国立研究所杜癌症，法新社的国立德拉RECHERCHE和地区阿尔萨斯。 彼得拉内韦一直支持基金会倒拉RECHERCHEMédicale。目前，他是一个因诺琴蒂基金会意大利语TECNOLOGIA（IIT）的奖学金的获得者。出版费用由Neurex网络支持（TriNeuron - 计划区域间四上莱茵省）

1，样品采集<ol><li>分离半粘合施耐德2细胞（1-5×10 6个细胞的平均值），通过移液并通过离心（100×g离心1分钟）收获它们。如果在体外培养的贴壁细胞，trypsinize他们在标准条件下，或者直接从板上将它们收集到的RNA提取试剂的便利量，用细胞刮刀的帮助。 </li><li>对于胚胎收集，成长果蝇株苍蝇笼子里，让胚胎积聚在产蛋板。在蒸馏水画笔（DH 2 O） 的胚胎擦拭掉，丢弃杂物用筛子过滤，冲洗和一小瓶48收回胚胎。 </li><li>作为一种替代技术，手动解剖组织/器官。在立体显微镜下（放大20倍）隔离腹果蝇睾丸（在piRNA基因分析的优惠制度）的磷酸盐缓冲液（PBS），用解剖针或镊子作为VISualized沙利文等 49。 </li><li>杵匀化后的0.5容积RNA提取试剂瘾样本。 </li></ol> 2，RNA提取/分析<ol><li>按照厂家的说明书，通过商业化的试剂进行RNA分离。 小心！RNA提取试剂通常毒性和刺激性。可替换地，应用的蛋白酶K的基于协议的RNA分离50，如下所示: <ol><li>样品稀释到400微升停止混合，并在室温下孵育15分钟。 </li><li>回收水溶性组分通过标准的苯酚:氯仿:异戊醇和氯仿提取并用乙醇（EtOH中）沉淀/洗涤。空气干燥样品重悬成焦碳酸二乙酯（DEPC）处理双蒸（DD）H 2 O </li></ol></li><li>评估紫外分光光度法测定RNA浓度。也保证了RNA的纯度高，接近2.0，BAS版上的260/280的阅读。 </li><li>检查RNA制备的变性琼脂糖凝胶如下: <ol><li>在一个干净的烧杯中，溶解1.2克琼脂糖82毫升DEPC-DDH 2 O，在不断搅拌下。煮至完全溶解。 </li><li>允许凝胶溶液冷却下来;当温度达到60℃，加上10×4吗啉酸10毫升（MOPS）缓冲液1X终浓度（FC）和8毫升37甲醛3％至％FC。 小心！甲醛是一种致癌物质。 </li><li>倒胶至水平电泳槽。使凝胶的发动机罩下固化至少1小时。下面凝固，淹没凝胶进入公开资讯观测站1X。 </li><li>分装2微克RNA​​样品和1微克天梯（使用一种RNA标记时，准确的评估需要）。加入3倍体积的琼脂糖样染料（ALD）的RNA和阶梯。热火在70℃下5分钟，立即在冰凉凉的。 加利福尼亚UTION！ALD的内容（溴化乙锭和甲酰胺，见材料）的诱变剂和腐蚀性，分别为。 </li><li>装入样品和运行凝胶为50 V与偶尔的缓冲回收约1小时。检查用UV可视化或凝胶成像RNA的分离模式。识别相应于28S和18S个rRNA和tRNA的到离散的条带（参见图1）。 </li></ol></li><li>继续北检测，或将RNA在-80℃。 </li></ol> 3，变性聚丙烯酰胺凝胶的制备<ol><li>用于该分级步骤一个小型垂直电泳仪（板尺寸长约8厘米×9厘米，梳厚度0.75mm）组合。 </li><li>组装清洁眼镜一起在试镜架。 </li><li>制备将10ml 10％丙烯酰胺/双丙烯酰胺（19:1）在MOPS 1X溶液，7M尿素。立即浇注之前，加入100微升10％过硫酸铵及10微升TEMED，并快速混合的溶液。 小心！丙烯酰胺具有神经毒性和致癌物质。 </li><li>倒入玻璃板之间的凝胶和仔细地插入以及梳，以避免气泡。让凝胶聚合​​在RT使用前至少45分钟。另外，存储凝胶的O / N，包裹在水中浸泡滤纸和保鲜膜密封。 </li></ol> 4，样品制备<ol><li>等分0.5-20总RNA为各样品的微克。如果体积超过3-4微升，真空干燥该样品。 </li><li>对于尺寸确定，并行运行的放射性低的范围梯等分（20-30 CPS）来样订做，作为标记（见4.3和4.4的准备）。在并行处理以样品，如在步骤4.5中描述。 </li><li>排行榜:设置使用0.1微克10个基点，阶梯的磷酸盐正向反应。在37℃下30分钟，使反应停止，加入乙二胺四乙酸（EDTA）以20mM的FC。 </li><li>通过乙醇沉淀/洗衣机，空气干燥和净化悬浮在DDH 2 O（约100微升）。 小心！32 P是一个放射源。 </li><li>向每个样品中添加的丙烯酰胺蓝色染料（ABD）的等体积。通过加热变性，在80℃下进行5分钟并在冰上冷却直到加载。 小心！甲酰胺（在ABD内容）是腐蚀性的。 </li></ol> 5，电泳分级<ol><li>从铸造支撑取出玻璃板并正确组装它们中的电泳单元。以足够的电泳缓冲液（RB）填充内和外室。 </li><li>轻轻取出梳子和运行前，在200V的凝胶10分钟。装载样品前，洗去从井尿素存款通过注射器喷出一些包。 </li><li>使用平头从步骤4.5在每个孔的底部仔细分层样本。检查阳极和阴极连接，以避免倒跑。后样品进入凝胶在100伏5分钟，我现象越来越多的电压200V。 </li><li>对于约7厘米长的分馏，45分钟，长期来看是足够的。避免溴酚蓝（在ABD跟踪颜料）溢出的胶。 </li><li>可视化的凝胶分级分离的长的RNA通过溴化乙锭（EtBr的）染色，作为一个可选的步骤: <ol><li>切开2厘米的凝胶的顶部，并简要冲洗在DDH 2 O的片</li><li>孵育凝胶切片在室温下进行15分钟，在RB，EtBr的0.5微克/毫升的FC温和正遨游。 </li><li>脱色在RB中的凝胶15分钟，可视化长个rRNA通过UV照射或凝胶成像。检查RNA的装载和质量（ 见图1，面板7）。 小心！溴化乙锭是一种诱变剂。 </li></ol></li><li>同时继续进行印迹含有小RNA种类的凝胶的下部。 </li></ol> 6，凝胶印迹<ol><li>切成6片吸墨纸以适合印迹区域的和等效的一块unchargeð尼龙膜。挫伤张纸张，薄膜和在RB 2印迹垫。请务必反复挤压他们的RB完全浸泡垫。 </li><li>除去从该装置中的凝胶并用抹刀​​的平均开眼镜。用干净的切割，除去孔内。将湿滤纸纸张上胶，轻轻抬不起来。 </li><li>将尼龙膜在凝胶上的自由表面。标志着一个角落里按装样以定向筛选。完成"三明治"（3纸片每边）。卷上的印迹表面的塑料棒，以消除任何可能的气泡。 </li><li>将两个印迹垫，然后之间的杂交，在杂交盒中。组装纸盒的印迹模块中，充满RB室，并检查是否有正确的方向（尼龙膜必须凝胶和正端之间留）。 </li><li>在传输20 V 20分钟。注:为确保均匀的条件下，经过10分钟打开印迹模块和R完成转接之前otate夹心180°。 </li></ol> 7，交联膜<ol><li>制备6毫升新鲜交联溶液（XLS）。饱和一块10厘米×10公分（大于尼龙膜）一张吸水纸，XLS， 小心！盐酸（XLS的一个组成部分）具有很强的腐蚀性。 </li><li>拆下的印迹，并将膜在湿3MM滤纸。注意:RNA必须不与饱和纸直接接触。将两块玻璃板之间的过滤器和纸，敷在萨兰换行。 </li><li>加热膜在60℃2小时，接着是DDH 2 O洗涤。储存在-20°C或进行杂交。 </li></ol> 8，膜预杂交<ol><li>如果装载的RNA已检查（可选步骤5.5），直接进行杂交。否则，切有1厘米的尼龙过滤器的顶部，以避免探针跨hybridizations至分馏长的RNA。 </li><li>预热在37℃10毫升杂交液（HS）的。变性1毫克鲑鱼精子在95℃下5分钟，在冰上冷却并添加到HS。 </li><li>在杂交炉中孵育协过滤器在37℃至少1小时，下旋转。检查过滤器的RNA的侧面爆头。 </li></ol> 9探针合成<ol><li>设立磷酸正向反应在10皮摩尔的反义寡核苷酸，如4.3节。 </li><li>加入5倍体积的Tris-EDTA-NaCl的（TEN）的缓冲液来进行反应和纯化来自未掺入的核苷酸的G-25葡聚糖凝胶柱的标记的引物通过凝胶排阻色谱法（或也通过乙醇沉淀）。探头活性可以通过液体闪烁计数器进行评估。 </li></ol> 10膜杂交<ol><li>更换用尽HS采用了新的10毫升一份（准备和补充如上所述）。热火在探头95℃下进行10分钟，在冰上冷却并添加到新的HS。孵育过滤器在37℃开。 </li></ol> 11膜的清洗<ol><li>恢复的杂交溶液中，它在-20℃下储存在放射性屏蔽盒。 </li><li>在冲洗洗涤缓冲液（WB）过滤器彻底清洗它在室温下10分钟。 </li><li>用盖革 - 米勒探测器为在滤波器的角落检查剩余放射性。当背景辐射大约是5厘，进入膜的论述。 </li></ol> 12，信号检测<ol><li>暴露在放射自显影胶片或在分子成像仪屏幕上的过滤器。揭示了照相处理或数字转换信号。 </li><li>分析由专门的量化软件（ImageJ的或Optiquant）的谱带强度。 </li></ol> 13，剥膜<ol><li>要删除基色信号，淹没膜在500毫升煮沸剥离液，然后在室温下孵育1分钟。前进到新的（预）杂交或存储滤波器在-20℃。 </li></ol>

The authors declare that they have no competing financial interests.

虽然我们的阅历相互交织，通过它的RNA调节神经的升值​​不断增加，基于RNA的设计电路影响神经干细胞生物学，神经细胞的分化/功能集成，神经病变和癌症的发展机理的影响尚未被探索。这样的网络通过王国的维护使得遗传系统的潜力作为果蝇有助于揭开未开发的细胞途径，其中短的非编码RNA和转录因子被认为是主要的分子球员。在此视图中，候选小分子RNA表达水平的概要是propaedeutic到...

RNA生物化学经历了惊人的进步，在过去的1年。我们的基因表达的对照RNA的潜在的理解已经爆裂通过强大的非编码核糖调节2的识别，通过新颖的基于RNA的调节机制3,4的发现和利用已公知的转录后事件更深的表征5。总之这些研究使RNA的生物学大大使场景，成为了目前的科学景观的一个主要研究课题。特别是近年来，我们越来越的"RNA世界"的分子神经生物学6，在现代生命科学中最具活力的研究领域之一的普遍冲击感。在过去的一个世纪的最后十年，整体科学的情景已经被彻底改变了小调控RNA 9,10的RNA干扰7,8的发现和特别是关于微小RNA 11，内源性表达牵连的几乎所有细胞功能基因表达的多效性和组合调节控制的小非编码RNA。 近10年后的miRNA最初发现在秀丽隐杆线虫由安布罗斯和Ruvkun的实验室，重新关注被拒绝到外地时，在果蝇和人类细胞以及12-15确定了高数量的miRNA。从那时起，这要归功于灵活的转基因方法， 果蝇已经站在了一个宝贵的生物背景的深入研究miRNA的生物合成和活性。 果蝇的miRNA揭示了不同的功能的昆虫特异性或进化上保守的过程，老化代谢，信号转导通路跨越，行为，当然，神经发生。沿着这个方向，我们最近推出了有趣的合作一个新的链接16rrelation主基因GCM /滑翔和RNA的通路之间发生。飞转录因子GCM /滑翔17-19构成的细胞命运的决定因素，这决定了胶质细胞与神经元命运的选择多能飞的神经前体20一个独特的例子。二十年对这个话题很长的研究已经清楚地强调了基因表达调控多和重叠的投入发生了融合GCM /滑行 21-28建立所需的神经发育过程中神经的平衡和神经胶质同行之间的微妙比例的阈值水平。 我们发现，通过DMEL-miR279目标，监管是进一步控制水平有助于转录后微调的GCM /滑动式16。从全球来看，这些研究都行需要具体的方法改进:沿几年，一些技术最初开发分析TRADitional的RNA被转化为定量小的非编码RNA，等作为RNA酶保护测定，cDNA芯片29-31，实时PCR的方法32-35和测序36,37。在另一边，技术方法校准助长了持续进展的领域。 Northern印迹法（NB，或RNA凝胶印迹法）构成了一个有代表性的实例:它主要用于型材RNA的积累，因为它确保了表达水平的定量和尺寸确定。然而，该方法的特性差的灵敏度被限制时，它是被应用到低丰度的基因表达细调谐器，像短的RNA。甲不利后果是大量的总RNA，这使得难以应用到特定的生物样品的要求。对于这样的理由，小分子RNA检测特定NB变种已经发展38-40:我们采取了一种改进的NB PROC的优势edure 41（ENB，增强Northern印迹），同时阐明DMEL-miR279和GCM /滑翔之间的上述相互作用。 此方法依赖于基于碳二亚胺衍生物的活性的化学交联步骤[1 - 乙基 - 3（3-二甲基氨基丙基）碳化二亚胺，EDC]固定核酸到固体支持物。碳二亚胺是已知催化的酰胺键的活化羧基或磷酸基团和胺基团42之间形成一个多功能的交联剂。此属性可通过其单磷酸5&#39;-羟基基团被利用共价耦合的小分子RNA，以氨基基团在尼龙膜的表面上。所得的附接结构增加了固定化的核酸的可访问性，反过来，探针-靶杂交，这导致显着的检测增强43的效率。 该技术假设特定relevancE在果蝇的分子生物学研究，通过这些新颖独特的类小非编码RNA的产生是新兴的44。在这些中，rasiRNAs 45,46构成PIWI相互作用RNA（了piRNA 47），参与序列特异性基因沉默的特异性亚型。此方法的执行细节中充分描述和可视化在下文中，相对于微小RNA DMEL-miR279和DMEL-miR286的分析，并在第一时间，有rasiRNA，rasi4的。我们推走极端此方法，该方法允许我们从RNA（小于1微克）的最小量的揭示表达很差目标。

<table><tbody><tr><td>FINAL COMPOSITION/NAME</td><td>COMPANY</td><td>CATALOGUE (Stock)</td><td>COMMENTS</td></tr><tr><td colspan="4">GENERAL REQUIREMENT</td></tr><tr><td>Centrifuge, 5418</td><td>Eppendorf</td><td>5418 000.017</td><td></td></tr><tr><td>Decontaminant, RNase Away</td><td>Sigma-Aldrich</td><td>83931</td><td>Apply on glass/plasticware, wipe and rinse</td></tr><tr><td>RNase inhibitor, DEPC</td><td>Sigma-Aldrich</td><td>D5758</td><td>Hazardous //// 1609-47-8</td></tr><tr><td colspan="4">SAMPLE PREPARATION</td></tr><tr><td>Stereo Microscope, M60</td><td>Leica&amp;nbsp;</td><td></td><td></td></tr><tr><td>1X PBS Buffer</td><td></td><td></td><td></td></tr><tr><td>137 mM NaCl</td><td>Sigma-Aldrich</td><td>S3014</td><td>7647-14-5&amp;nbsp;&amp;nbsp;</td></tr><tr><td>2.7 mM KCl</td><td>Sigma-Aldrich</td><td>P9541</td><td>7447-40-7&amp;nbsp;</td></tr><tr><td>10 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S3264</td><td>7558-79-4&amp;nbsp;</td></tr><tr><td>1.8 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P9791</td><td>7778-77-0&amp;nbsp;</td></tr><tr><td colspan="4">RNA EXTRACTION/ANALYSIS</td></tr><tr><td>UV-Vis Spectrophotometer</td><td>Thermo Scientific</td><td>Nanodrop 2000</td><td></td></tr><tr><td>Gel Imager, ChemiDoc XRS+</td><td>Biorad</td><td>170-8265</td><td></td></tr><tr><td>Horizontal Electrophoresys, Mini-Sub Cell GT</td><td>BioRad</td><td>170-4487EDU</td><td></td></tr><tr><td>RNA extraction reagent, TRIzol Reagent</td><td>Life Technologies</td><td>15596026</td><td>Hazardous</td></tr><tr><td>PCA (25:24:1), 1:1 for extraction</td><td>Sigma-Aldrich</td><td>77617</td><td>136112-00-0</td></tr><tr><td>Chlorophorm, 1:1 for extraction</td><td>Sigma-Aldrich</td><td>C2432</td><td>67-66-3&amp;nbsp;&amp;nbsp;</td></tr><tr><td>EtOH, 2.5 vol. for precipitation, 70% for washing</td><td>Sigma-Aldrich</td><td>59844</td><td>64-17-5&amp;nbsp;</td></tr><tr><td>Gene Ruler 1Kb DNA Ladder</td><td>Thermo Scientific</td><td>SM0311</td><td></td></tr><tr><td>Stop Mix</td><td></td><td></td><td></td></tr><tr><td>300 mM NaOAc, pH 5.5</td><td>Sigma-Aldrich</td><td>S2889</td><td>127-09-3</td></tr><tr><td>1x RSB Solution</td><td></td><td></td><td></td></tr><tr><td>2% sodium dodecyl sulfate (SDS)</td><td>Sigma-Aldrich</td><td>71736</td><td>Never cool down SDS to avoid&amp;nbsp; precipitation //// 151-21-3</td></tr><tr><td>2 mg/ml Proteinase K</td><td>Roche Applied Science</td><td>0-3115828001</td><td>EC 3.4.21.64 9</td></tr><tr><td>10X Reticulocyte Standard Buffer (RSB)</td><td></td><td></td><td></td></tr><tr><td>100 mM Tris-Cl, pH 7.4</td><td>Sigma-Aldrich</td><td>T5941</td><td>1185-53-1&amp;nbsp;&amp;nbsp;</td></tr><tr><td>100 mM NaCl</td><td>Sigma-Aldrich</td><td>S3014</td><td>7647-14-5&amp;nbsp;&amp;nbsp;</td></tr><tr><td>30 mM MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M8266</td><td>7786-30-3</td></tr><tr><td>Denaturing Agarose Gel</td><td></td><td></td><td></td></tr><tr><td>1.2% Agarose</td><td>Sigma-Aldrich</td><td>A9539</td><td>Let the gel solidify for at least 1 hr //// 9012-36-2</td></tr><tr><td>10X MOPS Buffer</td><td></td><td></td><td></td></tr><tr><td>0.2 M MOPS sodium salt, pH 7</td><td>Sigma-Aldrich</td><td>M9381</td><td>Do not confuse MOPS sodium salt with MOPS //// 71119-22-7</td></tr><tr><td>Agarose Loading Dye</td><td></td><td></td><td></td></tr><tr><td>1x MOPS Buffer</td><td></td><td></td><td></td></tr><tr><td>3% formaldehyde</td><td>Sigma-Aldrich</td><td>F8775</td><td>Hazardous //// 50-00-0</td></tr><tr><td>50% formamide</td><td>Sigma-Aldrich</td><td>47670</td><td>Hazardous //// 75-12-7</td></tr><tr><td>Ethidium bromide (EtBr) 30 &amp;mu;g/ml</td><td>Sigma-Aldrich</td><td>E1510</td><td>Hazardous //// 1239-45-8</td></tr><tr><td>Bromophenol blue&amp;nbsp;</td><td>Sigma-Aldrich</td><td>B0126</td><td>115-39-9&amp;nbsp;</td></tr><tr><td colspan="4">SMALL RNA FRACTIONATION</td></tr><tr><td>Flat gel loading tips</td><td>Life Technologies</td><td>LC1002</td><td></td></tr><tr><td>Savant SpeedVac Concentrator</td><td>Thermo Scientific&amp;nbsp;</td><td>DNA120</td><td></td></tr><tr><td>Vertical Electrophoresis, Mini-PROTEAN Tetra Cell&amp;nbsp;</td><td>Biorad</td><td>165-8000</td><td></td></tr><tr><td>Denaturing Acrylamide Gel</td><td></td><td></td><td></td></tr><tr><td>10% acrylamide/bis-acrylamide (19:1)</td><td>Sigma-Aldrich</td><td>A3449</td><td>Hazardous. Let the gel polymerize for 45 min before use</td></tr><tr><td>1x TBE Buffer (or 1x MOPS Buffer)</td><td></td><td></td><td></td></tr><tr><td>7 M urea</td><td>Sigma-Aldrich</td><td>U6504</td><td>57-13-6</td></tr><tr><td>0.1% ammonium persulfate</td><td>Sigma-Aldrich</td><td>215589</td><td>7727-54-0&amp;nbsp;</td></tr><tr><td>0.1% TEMED</td><td>Sigma-Aldrich</td><td>T9281</td><td>110-18-9</td></tr><tr><td>Gene Ruler Ultra Low Range DNA Ladder (10 bp-step)</td><td>Thermo Scientific</td><td>SM1211</td><td>Label 0.1 &amp;mu;g, as described in the &quot;probe labeling&quot; reaction</td></tr><tr><td>Acrylamide Blue Dye&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>50% formamide</td><td>Sigma-Aldrich</td><td>47670</td><td>Hazardous //// 75-12-7</td></tr><tr><td>Bromophenol blue</td><td>Sigma-Aldrich</td><td>B0126</td><td>115-39-9&amp;nbsp;</td></tr><tr><td>Running Buffer (RB)</td><td></td><td></td><td></td></tr><tr><td>1x MOPS Buffer&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Alternative RB: 1x TBE Buffer</td><td></td><td></td><td></td></tr><tr><td>1x TBE Buffer</td><td></td><td></td><td></td></tr><tr><td>5X TBE</td><td></td><td></td><td></td></tr><tr><td>445 mM Tris-base</td><td>Sigma-Aldrich</td><td>T1503</td><td>77-86-1&amp;nbsp;</td></tr><tr><td>445 mM boric acid</td><td>Sigma-Aldrich</td><td>B7901</td><td>10043-35-3</td></tr><tr><td>20 mM EDTA</td><td>Sigma-Aldrich</td><td>EDS</td><td>60-00-4&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Gel Staining Solution</td><td></td><td></td><td></td></tr><tr><td>Ethidium bromide 0.5 &amp;mu;g/ml</td><td>Sigma-Aldrich</td><td>E1510</td><td>Hazardous //// 1239-45-8</td></tr><tr><td>1x RB</td><td></td><td></td><td>Stain for 15 min with EtBr, destain for 15 min w/o EtBr</td></tr><tr><td colspan="4">BLOTTING</td></tr><tr><td>3MM Whatman paper</td><td>Sigma-Aldrich</td><td>Z270849</td><td></td></tr><tr><td>Neutral Nylon Membrane, Hybond NX</td><td>GE Healthcare Life Science</td><td>RPN203T</td><td>Photosensitive</td></tr><tr><td colspan="4">CROSSLINKING</td></tr><tr><td>Crosslinking Solution (XLS)</td><td></td><td></td><td></td></tr><tr><td>1% 1-methylimidazole (v/v)</td><td>Sigma-Aldrich</td><td>336092</td><td>Always prepare a fresh aliquot of XLS //// 616-47-7</td></tr><tr><td>12.5 mM HCl</td><td>Sigma-Aldrich</td><td>H1758</td><td>Hazardous //// 7647-01-0</td></tr><tr><td>3.1% EDC (w/v)</td><td>Sigma-Aldrich</td><td>3450</td><td>25952-53-8&amp;nbsp;&amp;nbsp;</td></tr><tr><td colspan="4">(PRE)-HYBRIDIZATION</td></tr><tr><td>Liquid Scintillation Counter&amp;nbsp;</td><td>Beckmann</td><td>LS 6500</td><td></td></tr><tr><td>Sheared Salmon Sperm, 0.1 mg/ml&amp;nbsp;</td><td>Life Technologies</td><td>AM9680</td><td></td></tr><tr><td>rasi4 anti-sense probe</td><td></td><td></td><td>5&#039; CGGUGUUCGACAGUUCCUCGGG -3&#039;</td></tr><tr><td>5s-rRNA anti-sense probe</td><td></td><td></td><td>5&#039;-CAACACGCGGTGTTCCCAAGCCG-3&#039;</td></tr><tr><td>Dmel-miR279 anti-sense probe</td><td></td><td></td><td>5&#039;-TTAATGAGTGTGGATCTAGTCA-3&#039;</td></tr><tr><td>Dmel-miR286 anti-sense probe</td><td></td><td></td><td>5&#039;-AGCACGAGTGTTCGGTCTAGTCA-3&#039;</td></tr><tr><td>Purification Kit, Illustra MicroSpin G-25 Columns</td><td>GE Healthcare Life Science</td><td>27-5325-01</td><td></td></tr><tr><td>(Pre)-Hybridization Solution (HS)</td><td></td><td></td><td></td></tr><tr><td>0.5% SDS</td><td>Sigma-Aldrich</td><td>71736</td><td>151-21-3&amp;nbsp;</td></tr><tr><td>5x Denhardt&#039;s Solution</td><td></td><td></td><td></td></tr><tr><td>20X SSPE</td><td></td><td></td><td></td></tr><tr><td>3.6 M NaCl</td><td>Sigma-Aldrich</td><td>S3014</td><td>7647-14-5&amp;nbsp;&amp;nbsp;</td></tr><tr><td>0.2 M sodium phosphate</td><td>Sigma-Aldrich</td><td>342483</td><td>7601-54-9&amp;nbsp;&amp;nbsp;</td></tr><tr><td>20 mM EDTA (pH 8.0)</td><td>Sigma-Aldrich</td><td>EDS</td><td>&amp;nbsp;60-00-4&amp;nbsp;&amp;nbsp;</td></tr><tr><td>50x Denhardt&#039;s Solution</td><td></td><td></td><td></td></tr><tr><td>1% Ficoll 400 (w/v)</td><td>Sigma-Aldrich</td><td>F2637</td><td>26873-85-8</td></tr><tr><td>1% Polyvinylpyrrolidone</td><td>Sigma-Aldrich</td><td>PVP40</td><td>9003-39-8</td></tr><tr><td>1% BSA</td><td>Sigma-Aldrich</td><td>A7906</td><td>9048-46-8&amp;nbsp;&amp;nbsp;</td></tr><tr><td>1X TEN Buffer</td><td></td><td></td><td></td></tr><tr><td>10 mM Tris-Cl (pH 8.0)</td><td>Sigma-Aldrich</td><td>T1503</td><td>77-86-1&amp;nbsp;</td></tr><tr><td>100 mM NaCl</td><td>Sigma-Aldrich</td><td>S3014</td><td>7647-14-5&amp;nbsp;&amp;nbsp;</td></tr><tr><td>1 mM EDTA (pH 8.0)</td><td>Sigma-Aldrich</td><td>EDS</td><td>60-00-4&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Probe Labelling</td><td></td><td></td><td></td></tr><tr><td>0.4 &amp;mu;M oligonucleotide</td><td></td><td></td><td></td></tr><tr><td>1.5 &amp;mu;l Ci/&amp;mu;l [&amp;gamma;-32P] ATP</td><td>Perkin Elmer</td><td>NEG002A</td><td>Hazardous //// 51963-61-2</td></tr><tr><td>1x T4 Polynucleotide Kinase Buffer</td><td>Biolabs</td><td>B0201</td><td></td></tr><tr><td>T4 Polynucleotide Kinase 0.5 units/&amp;mu;l</td><td>Biolabs</td><td>M0201</td><td>EC 2.7.1.78</td></tr><tr><td colspan="4">WASHING</td></tr><tr><td>Geiger Mueller Detectors</td><td>Canberra Industries</td><td>MCB2/CPS &amp;ndash; EM77021</td><td></td></tr><tr><td>Washing Buffer (WB), 2x SSPE</td><td></td><td></td><td></td></tr><tr><td>Stringent Washing Buffers</td><td></td><td></td><td></td></tr><tr><td>0.2x SSPE</td><td></td><td></td><td></td></tr><tr><td>2x SSPE, 0.5% SDS</td><td></td><td></td><td></td></tr><tr><td>0.2x SSPE, 0.5% SDS</td><td></td><td></td><td></td></tr><tr><td colspan="4">SIGNAL DETECTION</td></tr><tr><td>PharosFX Plus System</td><td>Biorad</td><td>170-9460</td><td></td></tr><tr><td>Image J</td><td>Freely available</td><td></td><td></td></tr><tr><td>Quantity One</td><td>Biorad</td><td></td><td></td></tr><tr><td>X-ray films , BioMax XAR</td><td>Sigma-Aldrich</td><td>F5888</td><td>Photosensitive</td></tr><tr><td>Developer for X-ray films</td><td>Sigma-Aldrich</td><td>P7042</td><td></td></tr><tr><td>Fixer for X-ray films</td><td>Sigma-Aldrich</td><td>P7167</td><td></td></tr><tr><td colspan="4">STRIPPING</td></tr><tr><td>Stripping Solution</td><td></td><td></td><td></td></tr><tr><td>10 mM Tris-HCl pH 8.8</td><td>Sigma-Aldrich</td><td>T1503</td><td>77-86-1&amp;nbsp;</td></tr><tr><td>5 mM EDTA</td><td>Sigma-Aldrich</td><td>EDS</td><td>60-00-4&amp;nbsp;&amp;nbsp;</td></tr><tr><td>0.1% SDS</td><td>Sigma-Aldrich</td><td>71736</td><td>Add SDS after boiling to avoid foaming //// 151-21-3</td></tr></tbody></table>

enhanced northern blot detection of small rna species in drosophila melanogaster

在过去的几十年目睹身边的基因表达调控机制的科学兴趣在RNA水平的爆炸。分子生物学的这个分支已经大大助长了非编码RNA作为主要参与者的转录后调控的发现。这样一种革命性透视一直伴随着与由强大的技术开发的仿形短的RNA表达触发，无论是在高通量水平（全基因组鉴定）或单候补分析（特定物种的稳态积累）。虽然国家的最先进的几种策略是目前可用于定量或可视化等逃离分子，RNA印迹分析仍然是分子生物学的RNA表达的即时和准确的评估资格的办法。它代表了对更复杂的，昂贵的技术，在许多情况下，应用程序的第一步，仍然是一个优先的方法来轻松地深入了解成RNA生物学。在这里，我们概述一个有效的协议（增强Northern杂交），用于检测果蝇弱表达的microRNA（或其他小调控RNA种类）全果蝇胚胎，人工解剖幼虫/成人组织或体外培养的细胞。是必需的RNA的一个非常有限的量，并利用材料从流式细胞仪，分离的细胞可以是也可以设想。

通过下面的文本中， 如图1中所描述和示意性地表示的整体过程中，我们评估不同来源的复杂的RNA样品的小RNA的表达。在图1中给出的实验中，RNA从果蝇施耐德2细胞通过蛋白酶K提取，检查其完整性分离（可选步骤:变性琼脂糖凝胶分离），并在双加载。凝胶的一半印迹在中性膜和化学交联的EDC;第二半被转移上带正电的尼龙过滤器，然后UV交联的（1.2×10 5个μJ/...

Watch this Scientific Journal Video about Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster at JoVE.com

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

本刊物的目的是可视化和探讨手术步骤，从果蝇的RNA提取的增强Northern杂交协议的果蝇胚胎，细胞和组织。这个协议是用于有效地检测小RNA种类是特别有用的。

增强Northern印迹检测小RNA种类的<em&gt;果蝇</em

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

enhanced-northern-blot-detection-small-rna-species-drosophila

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Biology

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

24.0K 次观看.  Institut de Génétique et de Biologie Moléculaire et Cellulaire. 本刊物的目的是可视化和探讨手术步骤，从果蝇的RNA提取的增强Northern杂交协议的果蝇胚胎，细胞和组织。这个协议是用于有效地检测小RNA种类是特别有用的。 

视频: Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

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

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

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

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

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

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

科研

遗传学

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and animals. Most miRNAs act as negative switches of gene expression targeting key genes. In plants, primary miRNAs (pri-miRNAs) transcripts are generated by RNA polymerase II, and they form varying lengths of stable stem-loop structures called pre-miRNAs. An endonuclease, Dicer-like1, processes the pre-miRNAs into miRNA-miRNA* duplexes. One of the strands from miRNA-miRNA* duplex is selected and loaded onto Argonaute 1 protein or its homologs to mediate the cleavage of target mRNAs. Although miRNAs are key signaling molecules, their detection is often carried out by less than optimal PCR-based methods instead of a sensitive northern blot analysis. We describe a simple, reliable, and extremely sensitive northern method that is ideal for the quantification of miRNA levels with very high sensitivity, literally from any plant tissue. Additionally, this method can be used to confirm the size, stability and the abundance of miRNAs and their precursors.

This method demonstrates use of the northern hybridization technique to detect miRNAs from total RNA extract.

用于植物微RNA检测和定量的RNA斑点分析

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...

生物化学

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

分离纯化果蝇</em磁性微球的排序>外周神经元

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

生物学

Single Nucleotide Polymorphism-sensitive FISH Detection of Locus-specific Ribosomal RNA Transcription in Drosophila melanogaster

In order to ensure sufficient ribosomal RNA (rRNA) is transcribed for ribosome function, genomes contain hundreds of tandem duplications of the sequences that encode rRNAs, composing regions called ribosomal DNA (rDNA) loci. Many organisms' genomes contain more than one rDNA locus distributed across different chromosomes, and rRNA may be transcribed from multiple rDNA loci or only a single locus. Changes in rRNA transcription sources often indicate ribosomal distress. However, the homogeneity of rRNA obstructs distinguishing if rRNA is transcribed from multiple or a single rDNA locus. Here, we describe a method that uses single nucleotide polymorphism-sensitive fluorescence in situ hybridization (SNP-FISH) to distinguish rRNA transcription between Drosophila melanogaster rDNA loci on the X and Y chromosomes. This method leverages locus-specific rRNA variants to enable easy detection of the source of rRNA transcription with single-cell resolution. This assay can be applied to any Drosophila cell type and may be adapted for use in other systems.

The protocol describes single nucleotide polymorphisms-sensitive fluorescence in situ hybridization (SNP-FISH) method to distinguish ribosomal RNA transcripts derived from the X or Y chromosome ribosomal DNA locus in Drosophila melanogaster.

黑腹果蝇基因座特异性核糖体 RNA 转录的单核苷酸多态性敏感 FISH 检测

In order to ensure sufficient ribosomal RNA (rRNA) is transcribed for ribosome function, genomes contain hundreds of tandem duplications of the sequences ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

RNAi的干扰双链RNA注射到果蝇胚胎

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

果蝇中单个 mRNA 分子的检测和定量

Studying Muscle Transcriptional Dynamics at Single-molecule Scales in Drosophila

Skeletal muscles are large syncytia made up of many bundled myofibers that produce forces and enable body motion. Drosophila is a classical model to study muscle biology. The combination of both Drosophila genetics and advanced omics approaches led to the identification of key conserved molecules that regulate muscle morphogenesis and regeneration. However, the transcriptional dynamics of these molecules and the spatial distribution of their messenger RNA within the syncytia cannot be assessed by conventional methods. Here we optimized an existing single-molecule RNA fluorescence in situ hybridization (smFISH) method to enable the detection and quantification of individual mRNA molecules within adult flight muscles and their muscle stem cells. As a proof of concept, we have analyzed the mRNA expression and distribution of two evolutionary conserved transcription factors, Mef2 and Zfh1/Zeb. We show that this method can efficiently detect and quantify single mRNA molecules for both transcripts in the muscle precursor cells, adult muscles, and muscle stem cells.

作者聚焦：研究果蝇肌肉组织中的 mRNA 空间分布 

Drosophila is a well-established model for studying key molecules that regulate myogenesis. However, current methods are insufficient to determine mRNA transcriptional dynamics and spatial distribution within syncytia. To address this limitation, we optimized an RNA fluorescence in situ hybridization method allowing the detection and quantification of mRNAs at a single-molecule scale.

研究果蝇单分子尺度的肌肉转录动力学

Skeletal muscles are large syncytia made up of many bundled myofibers that produce forces and enable body motion. Drosophila is a classical model to study ...

发育生物学

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

分离果蝇睾丸

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

增强Northern印迹检测小RNA种类的

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此视频中的章节

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

用于植物微RNA检测和定量的RNA斑点分析

分离纯化果蝇外周神经元

黑腹果蝇基因座特异性核糖体 RNA 转录的单核苷酸多态性敏感 FISH 检测

RNAi的干扰双链RNA注射到果蝇胚胎

研究果蝇单分子尺度的肌肉转录动力学

研究果蝇单分子尺度的肌肉转录动力学

研究果蝇单分子尺度的肌肉转录动力学

分离果蝇睾丸

全山RNA荧光