Name: analysis of the gap junction-dependent transfer of mirna with 3d-frap microscopy
Uploaded: 2017-06-19T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy at JoVE.com

德国联邦教育和研究部（FKZ 0312138A和FKZ 316159），梅克伦堡 - 西波美拉尼亚与欧盟结构基金（ESF / IVWM-B34-0030 / 10和ESF / IVBM-B35-0010 / 12），DFG（DA1296-1）和德国心脏基金会（F / 01/12）。此外，RD由罗斯托克大学医学中心（889001），DAMP基金会和BMBF（VIP + 00240）的FORUN计划支持。

根据罗斯托克大学医学中心动物护理的道德准则进行了涉及新生小鼠的本议定书中的所有步骤。 1.细胞培养皿和心肌细胞培养基的制备<ol><li>用PBS中的0.1％明胶包被细胞培养板，并在37℃下孵育4小时或在4℃下孵育过夜。取出明胶，使其在无菌层流空气流下干燥。 </li><li>准备由补充有10％胎牛血清（FBS）和1％青霉素/链霉素（P / S）的50mL的DMEM组成的细胞培养基。预热至37°C。 </li></ol> 2.新生儿心肌细胞的分离<ol><li>用无菌剪刀斩首新生儿小鼠（1-2天），沿胸骨打开胸部。使用镊子去除心脏，同时轻轻地将胸部按压在一起，并将心脏转移到含有冰冷HBSS的24孔板中（不含Ca 2+和Mg 2+ ）。 </li><li>使用无菌镊子去除非心脏组织和较大的血管。将清洁的心脏转移到含有冰冷HBSS的1.5mL管中。每管最多使用5颗心进行酶消化。 </li><li>用小剪刀将心脏夹碎成0.5至1毫米3片。 </li><li>使用1 mL微量移液管抽吸/添加HBSS，用HBSS洗涤切碎的心脏两次。加入酶前，彻底清除HBSS。 </li><li>对于新生儿心脏的酶消化，使用市售的试剂盒，并遵循制造商的方案（参见材料表 ）。每5分钟摇动含有切碎的心脏的管，同时用酶在37℃下孵育35分钟。 </li><li>将含有细胞培养基的未包被的细胞培养皿上的悬浮细胞悬浮1.5-2小时以允许非心肌细胞部分的粘附。如果高纯度重复此步骤需要心肌细胞。如果需要，进一步培养非心肌细胞部分。 注意:对于15个心脏的细胞悬浮液，可以在75cm 2的细胞培养瓶上进行预镀层步骤。通常，从一个新生儿心脏获得〜5×10 5个细胞。 </li><li>将上清液收集在15 mL锥形管中，并以300 x g离心10 min。将细胞重悬于1mL细胞培养基中。 </li><li>用Neubauer室血细胞计数器计数细胞，或使用等效方法。 </li><li>在密度为3×10 5个细胞/ cm 2的6孔板上平板分离的心肌细胞。在37℃和5％CO 2下孵育细胞过夜。 注意:任选地，细胞也可以在这一点被转染。然而，当在进行电穿孔之前将细胞培养一天时，观察到增加的活力。 </li></ol> 3.用荧光标记的miRNA进行转染<ol><li>预温水细胞培养基（参见步骤1.2）至37℃在水浴或等效装置中。 </li><li>在无RNase的无菌水中制备20μM的荧光miRNA储备溶液。 </li><li>制备含有90mM Na 2 HPO 4，90mM NaH 2 PO 4，5mM KCl，10mM MgCl 2和10mM琥珀酸钠的电穿孔缓冲液，并将pH调节至7.2;电穿孔缓冲液可以在-20°C储存几个月。 </li><li>使用0.05％胰蛋白酶从培养皿中分离细胞5分钟。通过添加细胞培养基灭活胰蛋白酶。 </li><li>用Neubauer室血细胞计数器计数细胞，或使用等效方法。将细胞以300xg离心10分钟。 </li><li>将细胞重悬于电穿孔缓冲液中以获得每100μL4×10 5个细胞的浓度。 </li><li> 将100μL细胞悬浮液与荧光miRNA（终浓度:0.25μM）在管中混合将混合物装入电穿孔试管。 <ol><li>根据细胞类型经验性地确定适当的miRNA数量。 </li></ol></li><li>使用"G-009"程序，使用电穿孔装置进行电穿孔（参见材料表 ）。 </li><li>加入500μL预热后的细胞培养液，将全细胞悬浮液（4×10 5个细胞）转移到4孔玻璃底部载玻片的孔中。在37℃，5％CO 2气氛中培养细胞1天。 注意:对于FRAP分析，细胞密度为〜80％是最佳的。标记的miRNA的转染应该仅使用电穿孔进行。由于标记的miRNA分子的均匀分布对于FRAP测量是有益的，因此不推荐基于试剂的转染。 </li></ol> 4.应用3D-FRAP显微镜（第3天） <ol><li>将显微镜孵化器预热至37°; C，并在FRAP测量前至少2小时开启共焦显微镜系统，建立热平衡并降低漂移的可能性。如果可能，保持5％二氧化碳气氛。 </li><li>将腔室载玻片插入平台样品架。 </li><li>使用1.4 NA油物镜（400倍放大）和561nm激光激发光在低激光功率下寻找转染的心肌细胞簇，检测范围为570-680nm。 注意:使用显微镜专用软件（参见"材料表" ）对FRAP设置，图像采集和分析进行了定义 。 </li><li> 激活"安装管理器"中的"z-Stack"，"时间序列"，"漂白"和"区域"按钮。 </li><li> 定义FRAP参数。 <ol><li>通过设置"帧大小"来定义"采集模式"菜单中的图像采集设置4;到512×512，"行步"为1，"扫描时间"为&lt;1秒。 </li><li>在"通道"菜单中，调整激光功率，偏移和增益设置，以从最小激光激发（ 例如，激光功率:1-5％）获得最大荧光;调整以避免强度饱和。将"针孔尺寸"设置为2μm。 </li><li>接下来，在"区域"菜单中，选择"ROI绘图工具"，并使用光标标记目标单元格，参考单元格和背景区域。如果需要，选择几个目标细胞进行光漂白。 </li><li>调整"漂白"菜单中的漂白设置（ 例如，迭代:9-14，光漂白激光功率:100％，图像采集间隔:60秒，周期:15）。定义3次初始扫描后的漂白开始。 </li><li>在"z-Stack"菜单中，根据单元格的厚度定义z-stack采集的限制。调整"数字of z层"至12 - 15。 </li><li>开始FRAP实验并记录荧光恢复。 注意:由于FRAP实验的设置取决于细胞类型，荧光染料和显微镜系统，最好进行试验性实验以确定FRAP的最佳参数。漂白应足以将初始荧光强度降低至少50％。通常，每30-60秒应记录荧光恢复，直到达到平台期。 </li></ol></li></ol>数据分析<ol><li> 创建所获得的z-叠层的最大投影并获得漂白的靶细胞，参考细胞和背景的荧光强度值。使用显微镜专用软件，点击"处理"→"最大强度投影"→选择文件→"应用"。 <ol><li>或者，使用等效图像分析工具（ <e例如，ImageJ）。 </li></ol></li><li> 将所有时间点的荧光强度数据从目标单元格，背景和参考单元复制到电子表格中。 <ol><li>从目标单元的强度值中减去参考单元的背景强度和强度，以校正在采集过程中产生的漂白。对每个时间点执行背景和参考单元校正。 </li><li>通过将每个值除以初始荧光，将校正的FRAP数据归一化为漂白前的初始荧光强度。 </li><li>为了获得FRAP曲线，通过从每个时间点的强度值中减去，将基线设置为漂白后立即的荧光强度。 </li></ol></li></ol>

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Y., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+delivery+of+miRNAs+for+cancer+therapy:+Challenges+and+strategies.">In vivo delivery of miRNAs for cancer therapy: Challenges and strategies.</a> Adv Drug Deliv Rev. 81C, 128-141 (2015).</li></ol>

作者宣称没有利益冲突。

<html> miRNA是细胞生理学中的关键参与者，并被证明通过使用G_s作为细胞间交换途径，作为信号分子11,12,22。目前的方案提出体外活细胞成像技术来表征使用荧光miRNA在细胞簇内的GJ依赖性穿梭。 该方案作为细胞模型系统在心肌细胞上开发。然而，如果电穿孔是miRNA转染的合适方法，则该方法可以应用于几种细胞类型。除了细胞间细胞交换，所提出的技术也适用于研究miRNA?...

小的非编码RNA是细胞基因调控的重要参与者。这些分子由与特异性靶mRNA结合的20-25个核苷酸组成，导致翻译的阻断或mRNA降解1,2 。由小RNA（例如miRNA和siRNA）进行的基因调控过程是许多不同物种中发现的高度保守的机制。特别地，miRNA分子对多种生理过程至关重要，包括增殖，分化和再生4,5 。此外，miRNA表达的失调归因于许多病理学障碍。相应地，miRNA已经被证明适合作为诊断的生物标志物和用作基因治疗的治疗剂6,7 </suP&gt;。 间隙连接（GJs）是两个相邻细胞的质膜中的专门的蛋白质结构，允许分子量高达1kD的扩散交换。已被证明对于组织发育，分化，细胞死亡和病理性疾病如癌症或心血管疾病8,9,10很重要 。已经描述了几个分子能够穿过GJ通道，包括离子，代谢物和核苷酸。有趣的是，还发现GJs为小RNA 11,12的细胞间移动提供了途径。因此，miRNA不仅可以在其产生的细胞内，而且在受体细胞内起作用。这突出了miRNA在细胞间信号转导系统中的作用。同时，数据显示该间隙连接细胞间通讯与miRNA功能密切相关。由于miRNA和GJs对组织稳态，病理学和诊断的重大影响，全面了解GJs的功能和miRNA的相关细胞间动力学有助于阐明基于miRNA的疾病的机制，并制定新的策略miRNA疗法。 根据间隙连接耦合的程度，miRNA分子在细胞间的转移可以是非常快速的过程。因此，需要一种允许可视化和量化这些调节信号分子的快速细胞间移动的方法。通常，已经应用流式细胞术和分子生物学技术来证明小RNA的穿梭11,12,13,14。然而，与FRAP微观相反复制，这些方法缺乏高时间分辨率，这是通过GJs分析miRNA的交换时强制的。此外，FRAP显微镜侵入性较小，因此代表了一种功能强大且新颖的活细胞成像技术，用于评估几种15,16,17型细胞中分子的GJ依赖性交换。 在这里，我们提出了一个详细的协议，描述了3D-FRAP应用于评估心肌细胞之间的miRNA穿梭。为此，用荧光标记的miRNA转染心肌细胞。用该miRNA标记的细胞被光漂白，并且以时间依赖的方式记录来自相邻细胞的间隙连接miRNA再流入。 FRAP实验的高时间分辨率提供了进行动力学研究以准确评估活细胞之间的miRNA和siRNA的细胞间转移的可能性。 Moreo由于小RNA可以通过具有高度不同动力学的不同机制进行交换，FRAP显微镜可以帮助澄清GJ在相应的穿梭过程18中涉及的程度。此外，3D-FRAP可用于研究GJ通透性的生理和病理学改变及其对小RNA转移的影响15,19。

<table><tbody><tr><td>neonatal NMRI mice</td><td>Charles River</td><td></td><td></td></tr><tr><td>Gelatin</td><td>Sigma Aldrich</td><td>G7041</td><td>0.1% solution in PBS, sterilized</td></tr><tr><td>PBS</td><td>Pan Biotech</td><td>P04-53500</td><td></td></tr><tr><td>Dulbecco&amp;acute;s modified medium</td><td>Pan Biotech</td><td>P04-03550</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td>100 U/mL, 100&amp;micro;g/mL</td></tr><tr><td>Fetal bovine serum</td><td>Pan Biotech</td><td>P30-3306</td><td></td></tr><tr><td>Cell culture plastic</td><td>TPP</td><td></td><td></td></tr><tr><td>Primary Cardiomyocyte Isolation Kit</td><td>Thermo Fisher Scientific</td><td>88281</td><td></td></tr><tr><td>HBSS</td><td>Thermo Fisher Scientific</td><td>88281</td><td>included in primary cardiomyocyte isolation kit</td></tr><tr><td>Trypan blue</td><td>Thermo Fisher Scientific</td><td>15250061</td><td></td></tr><tr><td>miRIDIAN Dy547 labeled microRNA Mimic</td><td>Dharmacon</td><td>CP-004500-01-05</td><td>dissolve in RNAse free water, stock solution 20&amp;micro;M</td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma</td><td>S3139</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Carl Roth</td><td>T877.2</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>P9333</td><td></td></tr><tr><td>Magnesium chloride</td><td>Serva</td><td>28305.01</td><td></td></tr><tr><td>Sodium succinate</td><td>Carl Roth</td><td>3195.1</td><td></td></tr><tr><td>0.05% Trypsin/EDTA solution</td><td>Merck</td><td>L2153</td><td></td></tr><tr><td>4-well- Glass bottom chamber slides</td><td>IBIDI</td><td>80827</td><td>coat with 0.1% gelatin solution</td></tr><tr><td>Amaxa Nucleofector II</td><td>Lonza</td><td></td><td>program G-009 was used for Electroporation&amp;nbsp;</td></tr><tr><td>LSM 780 ELYRA PS.1 system</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Excel software</td><td>Microsoft</td><td></td><td></td></tr><tr><td>&amp;alpha;-actinin antibody</td><td>Abcam</td><td>ab9465</td><td>dilution 1:200</td></tr><tr><td>Connexin43 antibody</td><td>Santa Cruz</td><td>sc-9059</td><td>dilution 1:200</td></tr><tr><td>goat anti-mouse Alexa 594 antibody</td><td>Thermo Fisher Scientific</td><td>A-11005</td><td>dilution 1:300</td></tr><tr><td>goat anti-rabbit Alexa 488 antibody</td><td>Thermo Fisher Scientific</td><td>A-11034</td><td>dilution 1:300</td></tr><tr><td>Connexin43 siRNA</td><td>Thermo Fisher Scientific</td><td>AM16708 ID158724</td><td>final concentration 250 nM</td></tr><tr><td>Near-IR Live/Dead Cell Stain Kit</td><td>Thermo Fisher Scientific</td><td>L10119</td><td></td></tr><tr><td>CellTrace Calcein Red-Orange</td><td>Thermo Scientific</td><td>C34851</td><td></td></tr><tr><td>DAPI nuclear stain</td><td>Thermo Scientific</td><td>D1306</td><td></td></tr></tbody></table>

analysis of the gap junction-dependent transfer of mirna with 3d-frap microscopy

小反义RNA（如miRNA和siRNA）在细胞生理学和病理学中起重要作用，而且可用作治疗几种疾病的治疗剂。开发miRNA / siRNA治疗新的创新策略是基于对基础机制的广泛了解。最近的数据表明，小细胞以间隙连接依赖的方式在细胞之间进行交换，从而在受体细胞中诱导基因调控作用。分子生物学技术和流式细胞仪分析通常用于研究miRNA的细胞间交换。然而，这些方法不提供高的时间分辨率，这在研究分子的间隙连接通量时是必需的。因此，为了研究miRNA / siRNA作为细胞间信号分子的影响，需要新的工具，这将允许在细胞水平上分析这些小RNA。本协议描述了ap光漂白后三维荧光恢复（3D-FRAP）显微镜的澄清，以阐明心脏细胞之间的miRNA分子的间隙连接依赖性交换。重要的是，这种直接和非侵入性的活细胞成像方法允许实时荧光标记的小RNA的间隙连接穿梭的可视化和定量，具有高的时空分辨率。通过3D-FRAP获得的数据证实了细胞间基因调控的新途径，其中小RNA作为细胞间网络内的信号分子。

在这里，我们提出3D-FRAP显微镜作为非侵入性技术研究新生儿心肌细胞荧光miRNA间隙连接穿梭。分离的心肌细胞显示典型的条纹α-肌动蛋白模式，并且沿着细胞 - 细胞边界包含大量的Cx43斑块（ 图1A ，白色箭头），这允许分子的高细胞间通量。通过显微定量α-肌动蛋白阳性细胞评估分离的心肌细胞的纯度。虽然培养物中存在一些非心肌细胞（α-肌动蛋?...

Watch this Scientific Journal Video about Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy at JoVE.com

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

在这里，我们描述了光漂白后三维荧光恢复（3D-FRAP）的应用，用于分析miRNA的间隙连接依赖穿梭。与通常应用的方法相比，3D-FRAP允许实时定量小RNA的细胞间转移，具有高的时空分辨率。

用3D-FRAP显微镜分析miRNA的间隙连接依赖性转移

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

analysis-gap-junction-dependent-transfer-mirna-with-3d-frap

Research

JoVE Journal

Biology

8.8K 次观看.  Reference and Translation Center for Cardiac Stem Cell Therapy (RTC). 在这里，我们描述了光漂白后三维荧光恢复（3D-FRAP）的应用，用于分析miRNA的间隙连接依赖穿梭。与通常应用的方法相比，3D-FRAP允许实时定量小RNA的细胞间转移，具有高的时空分辨率。 

视频: Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

科研

生物工程

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3&#39; UTR of the target mRNA is mediated by a 2&#8211;8 nucleotide seed sequence at the 5&#39; end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3&#39; UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3&#39; UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

基于生物素的下拉法对细胞 miRNAs mRNA 靶点的验证

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

遗传学

Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy

The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated miRNAs rather than the action of a single miRNA. The characterization of these specific miRNAs modules is a fundamental step in maximizing their use in therapy. This is extremely relevant because their combinatorial attributes can be practically exploited. Described here is a method to define a specific miRNA signature relevant to the control of oncogenic chromatin repressors in glioblastoma. The approach first defines a general group of miRNAs that are deregulated in tumors in comparison to normal tissue. The analysis is further refined by differential culture conditions, underscoring a subgroup of miRNAs that are co-expressed simultaneously during specific cellular states. Finally, the miRNAs that satisfy these filters are combined into an artificial polycistronic transgenes, which is based on a scaffold of naturally existing miRNA clusters genes, then used for overexpression of these miRNA modules into receiving cells.

Described here is a protocol for characterizing modules of biologically synergistic miRNAs and their assembly into short transgenes, which allows simultaneous overexpression for gene therapy applications.

胶质母细胞瘤中功能相关miRNA的特性及其工程在基因治疗的人工簇中

The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated ...

A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves different RNA-binding proteins in the nucleus. Mature miRNAs frequently mediate gene silencing, and this has become an important tool for comprehending post-transcriptional events. Besides that, it can be explored as a promising methodology for gene therapies. However, there is currently a lack of direct methods for assessing miRNA expression in mammalian cell cultures. Here, we describe an efficient and simple method that aids in determining miRNA biogenesis and maturation through confirmation of its interaction with target sequences. Also, this system allows the separation of exogenous miRNA maturation from its endogenous activity using a doxycycline-inducible promoter capable of controlling primary miRNA (pri-miRNA) transcription with high efficiency and low cost. This tool also allows modulation with RNA-binding proteins in a separate plasmid. In addition to its use with a variety of different miRNAs and their respective targets, it can be adapted to different cell lines, provided these are amenable to transfection.

We developed an intronic microRNA biogenesis reporter assay to be used in cells in vitro with four plasmids: one with intronic miRNA, one with the target, one to overexpress a regulatory protein, and one for Renilla luciferase. The miRNA was processed and could control luciferase expression by binding to the target sequence.

一种报告基因检测法，用于分析哺乳动物细胞中内旋微RNA成熟

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves ...

癌症研究

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

miRNA的目标使用的任务目标ID库的全基因组屏幕

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

生物学

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

模仿人类小分子RNA的LightSwitch萤光素酶检测系统使用3&#39;UTR记者构造的microRNA识别目标在贴壁细胞

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

小RNA分子的高效结扎的微小RNA定量高通量测序

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

基于探针实时荧光定量PCR途径的小分子RNA定量检测

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

检测miRNA靶在高通量的使用3&#39;LIFE分析

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.

用3D-FRAP显微镜分析miRNA的间隙连接依赖性转移

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高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidigm公司动态ArrayTM国际金融中心

基于生物素的下拉法对细胞 miRNAs mRNA 靶点的验证

胶质母细胞瘤中功能相关miRNA的特性及其工程在基因治疗的人工簇中

一种报告基因检测法，用于分析哺乳动物细胞中内旋微RNA成熟

miRNA的目标使用的任务目标ID库的全基因组屏幕

模仿人类小分子RNA的LightSwitch萤光素酶检测系统使用3'UTR记者构造的microRNA识别目标在贴壁细胞

小RNA分子的高效结扎的微小RNA定量高通量测序

基于探针实时荧光定量PCR途径的小分子RNA定量检测

检测miRNA靶在高通量的使用3'LIFE分析

微操作系统技术允许分析骨架调节器的形态发生动力学和营业额

用3D-FRAP显微镜分析miRNA的间隙连接依赖性转移

摘要

探索更多视频

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高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

基于生物素的下拉法对细胞 miRNAs mRNA 靶点的验证

胶质母细胞瘤中功能相关miRNA的特性及其工程在基因治疗的人工簇中

一种报告基因检测法，用于分析哺乳动物细胞中内旋微RNA成熟

miRNA的目标使用的任务目标ID库的全基因组屏幕

模仿人类小分子RNA的LightSwitch萤光素酶检测系统使用3'UTR记者构造的microRNA识别目标在贴壁细胞

小RNA分子的高效结扎的微小RNA定量高通量测序

基于探针实时荧光定量PCR途径的小分子RNA定量检测

检测miRNA靶在高通量的使用3'LIFE分析

微操作系统技术允许分析骨架调节器的形态发生动力学和营业额

高通量微RNA分析：优化复纳升秤定量RT - PCR Fluidigm公司动态ArrayTM国际金融中心