这项工作得到了国家卫生研究院 (NIH)、DA024558、DA30896、DA033892、DA021471、AI22960 和 MD007586 (C.D.) DA037779 的部分支持。这项工作还得到了 RCMI 赠款 G12MD007586、范德比尔特 CTSA 补助金 UL1RR024975、Meharry 翻译研究中心 MeTRC 赠款 (CTSA U54/nih、RR026140/国立卫生研究院 NCRR 补助金和田纳西州艾滋病中心) 的支持。研究 (P30 AI110527)。

1. 转染 3 '-生物素化 miRNA
注: 在无菌层流罩内执行以下步骤。
<ol><li>种子 4 x 105–5 x 105 HEK-293T 细胞每井在2毫升完全 Dulbecco 的改良鹰培养基 (DMEM) [DMEM 补充10% 胎牛血清 (血清) 和1x 笔链球菌抗生素]。培养细胞在孵化器设置在37°c, 5% CO2隔夜。</li>
	<li>第二天, 检查在光显微镜下被镀细胞的健康和黏附。 注意: 确保细胞在继续下一步之前坚持并获得适当的形态学。HEK-293T 细胞通常准备转染 18–24 h 后电镀。</li>
	<li>在一个无菌的1.7 毫升离心管, 稀释 75 picomoles 生物素化 miRNA 200 µL 最小的基本介质。将这种混合物转移到另一种1.7 毫升离心管, 含有脂质体基转染试剂 (8 µL/井) 稀释200µL 的最小基本介质。完全混合, 但轻轻地, 通过吹打和孵化20–25分钟在室温允许形成转染复合物。</li>
	<li>用温和的吹打从6井培养板中取出旧介质, 用1.6 毫升新鲜制备的完整 DMEM (不需要1x 枝链球菌抗生素) 补充每一个井。</li>
	<li>使用校准良好的吸管将转染复合物 (从 1.3) 降为6井板中的细胞。在添加复合物的同时, 轻轻地旋转盘子, 以确保其均匀分布在板材上。 注意: 这一步对于实现高效转染非常关键, 必须谨慎和耐心地进行。</li>
	<li>培养细胞在37°c 和 5% CO2为至少 36 h。</li>
</ol>2. 链亲和素涂覆磁珠的制备-I。
<ol><li>并用重悬链亲和素磁珠彻底涡流。将30µL 的珠悬浮 (每样) 转移到2毫升核酸酶离心管。</li>
	<li>将包含珠悬浮的管放在磁珠分离器支架上 ("磁铁" 以后) 为2分钟。在确保珠子被拉到与磁铁接触的管子侧面后, 用微小心地取出上清液。</li>
	<li>添加100µL 的珠洗涤缓冲器 (10 毫米三氯 pH 7.5, 0.5 毫米 EDTA, 1 米氯化钠) 的珠子。把管子从磁铁上取下。漩涡为十五年代在室温下洗涤珠子。</li>
	<li>将含有珠子的管子放在磁铁上2分钟, 小心地除去并丢弃上清。</li>
	<li>重复步骤2.3 和 2.4, 总共三洗涤。最后洗完一步后, 从磁铁上取下管子。</li>
	<li>添加100µL 的 RNase 释放溶液 (0.1 米氢氧化钠, 0.05 米氯化钠) 的珠子, 混合在十五年代的涡流, 并在室温下孵化5分钟. 将含珠的管放在磁铁上2分钟, 并小心地除去并丢弃上清。</li>
	<li>重复步骤 2.6, 总共三次。</li>
	<li>添加珠泥沙溶液 (0.5 米氯化钠) 到珠, 漩涡十五年代, 并在室温孵育5分钟. 将悬浮珠放在磁铁上2分钟, 并小心地取出上清。</li>
	<li>添加200µL 的珠阻断溶液 (1 µg/µL BSA, 2 µg/µL 酵母 tRNA) 的珠子。在室温下由温和的涡流混合在一起十五年代。在多管旋转体上孵育16小时 (超晚) 4 摄氏度。</li>
</ol>3. 细胞裂解物的制备
<ol><li>在层流罩内用不育的细胞刮板来收割转染的 HEK-293T 细胞, 然后将每个样品转移到无菌的2毫升离心管中。</li>
	<li>用离心法在 1500 x g上并用重悬5分钟, 在1x 无菌 PBS (磷酸盐缓冲盐水) 中颗粒, pH 7.2。离心机再以 1500 x g为5分钟, 获得无残留介质的颗粒。立即将含有颗粒的管子浸入冰中。</li>
	<li>制备新鲜完整细胞裂解缓冲液 (150 毫米氯化钠, 25 毫米三氯, pH-7.5, 5mM, 0.5% IGEPAL, 60 U/毫升 Superase, 1x 蛋白酶抑制剂)。 注: 缺乏 IGEPAL、Superase 和蛋白酶抑制剂鸡尾酒的细胞裂解缓冲液可以提前制备, 并贮存在室温下。通过将上述补充剂添加到库存细胞裂解缓冲液中, 并将其储存在冰上, 准备一组全新的完整细胞裂解缓冲液。</li>
	<li>将260µL 的冰冷完整细胞裂解缓冲液添加到离心管中的每个样品中 (即,每个离心管都含有从6井板的一个井中收获的细胞), 并并用重悬细胞颗粒成均匀悬浮由吹打。</li>
	<li>用冻融法溶解细胞: 在-80 摄氏度的10–15中孵育试管。然后, 允许细胞在冰上解冻。</li>
	<li>离心机所产生的细胞裂解在 1.6万 x g 5 分钟的冷冻台式离心机设置为4摄氏度。</li>
	<li>将清除的细胞裂解液转移到冰上无菌的1.7 毫升离心管上。扔掉小球。 注: 清除的裂解液的最终体积应该是 ~ 240–250µL。</li>
	<li>将5M 氯化钠添加到已清除的裂解液中, 最后浓度为 1M, 并在冰上保持样品。</li>
</ol>4. 链亲和素磁珠的制备-II。
<ol><li>准备新鲜完整的下拉洗涤缓冲器 (10 毫米氯化钾, 1.5 毫米氯化镁2, 10 毫米三氯 pH 值 7.5, 5 毫米, 1 米氯化钠, 0.5% IGEPAL, 60 u/毫升 Superase, 1x 蛋白酶抑制剂鸡尾酒) 注: 不 IGEPAL、Superase 和蛋白酶抑制剂鸡尾酒的下拉式洗涤缓冲器库存, 可预先准备, 并存放在室温下。通过将上述补充剂添加到库存细胞裂解缓冲液中, 并将其储存在冰上, 准备一组全新的完整细胞裂解缓冲液。</li>
	<li>把装有准备好的珠子的管子 (从步骤 2.9) 放在磁铁上2分钟. 小心地取出并丢弃上清。</li>
	<li>添加150µL 的冰冷完整的下拉洗涤缓冲区的珠子。十五年代的旋涡, 在室温下孵化 30–60 s. 把管子放在磁铁上2分钟小心地取出并丢弃上清。</li>
	<li>重复步骤 4.3, 总共3次。</li>
	<li>并用重悬在300µL 的完整拉下洗涤缓冲器的珠子。</li>
</ol>5. 目标 mRNA-miRNA 配合物的下拉
<ol><li>将300µL 细胞裂解液 (从步骤 3.8) 转移到含有300µL 珠的离心管 (从步骤 4.5)。</li>
	<li>在室温下在 nutating 搅拌机上孵化混合物1小时。</li>
	<li>把管子放在磁铁上5分钟, 小心地取出并丢弃上清。</li>
	<li>添加300µL 的冰冷完整的下拉洗涤缓冲区的珠子。漩涡为十五年代在室温和安置管子在磁铁5分钟. 小心地去除和丢弃上清。</li>
	<li>重复步骤5.4 次。</li>
	<li>并用重悬在100µL 核酸酶水中的珠子, 在冰上孵化。</li>
</ol>6. 总 RNA 提取、cDNA 合成和 qPCR
<ol><li>使用适当的方法从悬浮珠子 (从步骤 5.6) 中提取总 RNA (见材料表)。并用重悬提取的总 RNA 在25µL 的核酸酶无水。用分光光度法测定 RNA 浓度和质量 (吸收率在 260/280)。准备50µL 的 RNA 的工作存货。</li>
	<li>在 triplicates 中, 使用适当的 cdna 合成试剂盒 (见材料表), 在最后体积为20µL (表 1) 中使用 50 ng 总 RNA (从步骤 6.1) 中进行 cdna 合成。然后, 根据表 2中描述的条件, 将 PCR 管加载到 qPCR 仪器中进行热循环。</li>
	<li>
使用 SYBR 绿色化学 (见材料表) 对 CDNA 进行 qPCR (从步骤6.2 中):
	<ol>
<li>在无菌条件下, 在96井清楚的底板上进行 triplicates 的所有 qPCR 反应。</li>
		<li>整除9µL 的反应混合物从 qPCR 主混合 (见表 3) 到每一个良好的板块。然后, 在每个井中添加1µL 的 cDNA, 以达到10µL 的最终体积. 用耐热 pcr 板封口机密封 pcr 板并将其加载到 qPCR 仪器中</li>
		<li>按照表 4中描述的条件执行热循环。</li>
		<li>将每个示例的表达式级别 (Ct 值) 规范化到被炒控件的表达式级别。使用这些值计算表达式中的折叠更改。</li>
	</ol>
</li>
</ol>

作者声明没有竞争的金融和非金融利益。

细胞 miRNAs mRNA 靶点的鉴定对于了解其调控功能具有重要意义。基于种子序列互补和保护目标序列19、20, 采用了几种计算工具对目标进行预测。虽然这些工具很有价值, 但它们可以产生高级别的误报和假阴性。因此, 这些预测结合了实验方法, 如 RISC 复合体的免疫沉淀和 miRNA 的拉下: mRNA 复合体, 以确认 miRNAs 对各自目标27,<sup class=...

MicroRNAs (miRNAs) 是小的非编码 rna, 负调节蛋白表达1。miRNA 的前体通过基因组的许多区域聚集, 最频繁地在基因间区域和蛋白质编码基因的内含子2,3。合成的 miRNAs 涉及转录 miRNAs 从 miRNA 编码基因4。miRNAs 进行序贯处理, 先在细胞核内, 然后在细胞质中生成单股 miRNAs4,5。随后, 成熟的 miRNAs 被纳入到 RNA 诱导的沉默复合体 (RISC): 一个 multimeric 蛋白-RNA 复合物, 其中包括 Argonaute 的蛋白质家族成员的目标识别4,5, 6,7。在 RISC 复杂的成熟 miRNAs 主要绑定到目标基因8,9,10的 3 ' UTR, 但也可以偶尔绑定在编码和 5 ' UTR 地区的 mRNA10,11.miRNA 与 mrna 序列的结合导致了平移沉默12,13,14 , 在某些情况下 mRNA 失稳15。由于单个 miRNA 可以针对许多基因, 这些调控分子参与几乎每一个细胞过程, 并有牵连在各种疾病情况下16,17,18。
详细了解 miRNAs 如何调节细胞通路需要识别目标基因。多个生物信息学平台可用于预测假定的 miRNA: mRNA 相互作用19,20。这些预测依赖于完美的沃森-克里克基配对的2–8核苷酸种子序列的 miRNA 和一个互补序列内的目标 mRNA4,21。此外, 这些工具生成 miRNA 的二级结构: mRNA 双工, 计算这种分子相互作用的热力学参数, 并显示跨物种的结合点的保护, 以提高目标的功能相关性预测。不幸的是, 这些工具也有限制预测假阳性目标在一个非常高的速度 (〜 27–70%)15,22。最重要的是, 这些在硅平台中无法识别 miRNAs 与目标23的非规范交互。因此, 这种预测分析往往与实验方法相结合, 以验证功能相关的目标。
已经开发了多种方法来实验验证 miRNA: mRNA 相互作用。使用 miRNA 模拟器、海绵和抑制剂的基因实验, 改变细胞 miRNAs 的水平, 为其对目标基因表达24、25、26的调控作用提供了线索。此外, 通过联合转染包含目标 mRNA 3 ' UTR 区域的克隆和 miRNA 模拟器或抑制剂进入细胞, 以记者为基础的化验, 提供了 miRNAs26的调节功能的证据。这些方法对于研究基因表达后转录调控的 miRNAs、转染效率和细胞 miRNA 水平变化的 pleotropic 效应是至关重要的, 这些遗传方法的主要局限性是23, 26。因此, miRNA 与靶的直接相互作用的补充生物化学方法, 可以更好地了解 miRNAs 的细胞功能。
研究 miRNA 与目标直接相互作用的一种广泛应用的方法是 RISC 复合体的免疫沉淀, 其次是复杂27、28、29、30 中的 mRNA 靶检测..最近, 还利用改进的 RISC 陷阱方法识别 miRNA 目标;它在 miRNA mrna 中间体中对目标进行稳定, 并对 mrna 靶点进行纯化。然而, 这些 methodsface 了 rna 和 rna 结合蛋白之间的非特异相互作用的内在挑战, 通常由细胞间隔31,32隔开。此外, 这些化验是依赖于存在的 AGO2 蛋白免疫沉淀的 RISC 复杂33。鉴于 AGO2 不是唯一的 argonaute 调解有效的 miRNA: mRNA 相互作用, 排除其他 argonautes 可能导致有偏见的结果34。因此, 要研究 miRNA 对 mRNA 的直接结合, 需要采取替代的策略。
在本报告中, 我们详细阐述了一个 miRNA 和它的目标 mRNA 直接相互作用的方法。首先, 3 ' 生物素化锁定核酸 (低噪声放大) miRNA 模仿者转染成哺乳动物细胞。然后, miRNA: 细胞裂解物中的 mRNA 复合体是用链亲和素包覆的磁性珠子捕获的。用 qPCR 量化了其互补 miRNA 的 mRNA 靶。

<table><tbody><tr><td>Cell line- HEK293T cells</td><td>ATCC</td><td>CRL-3216</td><td></td></tr><tr><td>Heat-inactivated Fetal bovine serum (Hi-FBS)</td><td>GIBCO/Thermofisher</td><td>10438-026</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM)</td><td>GIBCO/Thermofisher</td><td>11995-065</td><td></td></tr><tr><td>Phosphate buffered saline (PBS) (1x)</td><td>GIBCO/Thermofisher</td><td>20012-027</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)&amp;nbsp;</td><td>GIBCO/Thermofisher</td><td>25200-056</td><td></td></tr><tr><td>Penicillin-Streptomycin solution (100x)</td><td>Cellgro/Mediatech</td><td>30-002-CI</td><td></td></tr><tr><td>DNase&amp;nbsp;</td><td>Ambion</td><td>AM2238</td><td></td></tr><tr><td>Opti-MEM Reduced serum media</td><td>GIBCO/Thermofisher</td><td>3198-088</td><td></td></tr><tr><td>Liopfectamine 2000 Transfection reagent</td><td>Invitrogen/ Thermofisher</td><td>11668-019</td><td></td></tr><tr><td>Biotinylated hsa-miR-125b</td><td>Exiqon</td><td>339178/ID-27281622</td><td></td></tr><tr><td>Biotinylated scrambled control</td><td>Exiqon</td><td>479997-671/ID-714884</td><td></td></tr><tr><td>IGEPAL</td><td>Sigma-Aldrich</td><td>18896</td><td></td></tr><tr><td>Streptavidin Magnetic beads</td><td>Pierce</td><td>88816</td><td></td></tr><tr><td>Yeast tRNA</td><td>Invitrogen/Thermofisher</td><td>15401-011</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A2153</td><td></td></tr><tr><td>Potassium chloride (KCl) solution</td><td>Sigma-Aldrich</td><td>60142</td><td></td></tr><tr><td>Magnesium chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;) solution</td><td>Sigma-Aldrich</td><td>M1028</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Sigma-Aldrich</td><td>795429</td><td></td></tr><tr><td>Ethylenediamine tetraacetic acid, disodium salt (EDTA) solution, 0.5 M, pH 8.0</td><td>Sigma-Aldrich</td><td>E7889</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Sigma-Aldrich</td><td>43815</td><td></td></tr><tr><td>Superase</td><td>Ambion/Thermofisher</td><td>AM2694</td><td></td></tr><tr><td>Protease Inhibitor cocktail</td><td>Sigma-Aldrich</td><td>P8340</td><td></td></tr><tr><td>RNAse/DNAse free water</td><td>GIBCO/Thermofisher</td><td>10977-015</td><td></td></tr><tr><td>RNeasy extraction kit</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>iScript-Select cDNA synthesis kit</td><td>Biorad</td><td>170-8897</td><td></td></tr><tr><td>qPCR Primers&amp;nbsp;</td><td>Invitrogen/Thermofisher</td><td></td><td></td></tr><tr><td>iTaq-Universal SYBR green supermix</td><td>Biorad</td><td>172-5120</td><td></td></tr><tr><td>DynaMag-Spin Magnet</td><td>Invitrogen/Thermofisher</td><td>12320D</td><td></td></tr></tbody></table>

biotin-based pulldown assay to validate mrna targets of cellular mirnas

MicroRNAs (miRNAs) 是一类小非编码 rna, 后转录调节细胞基因表达。MiRNAs 绑定到 3 ' 未翻译的区域 (UTR) 的目标 mRNA, 以抑制蛋白质的翻译或在某些情况下导致 mRNA 退化。miRNA 对目标 mRNA 的 3 ' UTR 的绑定由 miRNA 5 ' 末端的2–8核苷酸种子序列介导。虽然 miRNAs 作为细胞调控分子的作用已经建立, 但与功能相关的目标基因的识别仍然是一个挑战。生物信息学工具被用来预测 3 ' UTR 基因中的序列, 作为 miRNA 绑定的潜在目标。这些工具也被用来确定这些序列在相关物种之间的进化守恒, 以预测功能作用。然而, 这些计算方法往往产生假阳性结果, 仅限于预测 miRNA 和 mRNA 之间的典型交互作用。因此, 测量 miRNA 对其 mRNA 靶的直接约束的实验程序是建立功能交互的必要条件。在本报告中, 我们描述了一种敏感的方法来验证细胞 miRNA miR-125b 和 3 ' UTR PARP-1 mRNA 之间的直接相互作用。我们制定了一个协议, 其中合成生物素化 miRNA 模仿被转染成哺乳动物细胞和 miRNA mRNA 复合体的细胞裂解物被拉下来与链亲和素涂层磁性珠。最后, 采用 qPCR 策略对下拉核酸复合体中的靶 mRNA 进行量化。

MiRNAs 通过绑定到目标基因来调节细胞过程。因此, 识别 mRNA 目标是理解 miRNA 功能的关键。在此, 我们详细阐述了一种识别细胞 miRNA mRNA 靶的方法。这个协议是从迩那里改编的。35利用生物素化低噪声的 miRNA 模拟到下拉靶 mRNA 的改进。基于低噪声的寡核苷酸模拟提高了靶结合的特异性, 由于改性核糖环的稳定性较高, 不含毒性效应36<...

Watch this Scientific Journal Video about Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs at JoVE.com

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

本报告描述了一种快速可靠的方法来验证细胞 miRNAs 的 mRNA 目标。该方法采用合成生物素化锁定核酸 (低噪声放大) miRNA 模拟捕获靶 mRNA。随后, 采用链亲和素包覆磁性微珠对靶 mRNA 进行 qPCR 聚合酶链反应的定量。

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; ...

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JoVE Journal

Genetics

13.7K 次观看.  Kalinga Institute of Industrial Technology University. 本报告描述了一种快速可靠的方法来验证细胞 miRNAs 的 mRNA 目标。该方法采用合成生物素化锁定核酸 (低噪声放大) miRNA 模拟捕获靶 mRNA。随后, 采用链亲和素包覆磁性微珠对靶 mRNA 进行 qPCR 聚合酶链反应的定量。

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

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3&#39; end. Reverse transcription and amplification of the transcripts are not required. Reporter probes contain a sequence of six color positions populated using a combination of four fluorescent colors. The four colors over six positions are used to construct a gene-specific color barcode sequence. Post-hybridization processing is automated on a robotic prep station. After hybridization, the excess probes are washed away, and the tripartite structures (capture probe-miRNA-reporter probe) are fixed to a streptavidin-coated slide via the biotin-labeled capture probe. Imaging and barcode counting is done using a digital analyzer. The immobilized barcoded miRNAs are visualized and imaged using a microscope and camera, and the unique barcodes are decoded and counted. Data quality control (QC), normalization, and analysis are facilitated by a custom-designed data processing and analysis software that accompanies the assay software. The assay demonstrates high linearity over a broad range of expression, as well as high sensitivity. Sample and assay preparation does not involve enzymatic reactions, reverse transcription, or amplification; has few steps; and is largely automated, reducing investigator effects and resulting in high consistency and technical reproducibility. Here, we describe the application of this technology to identifying circulating miRNA perturbations in irritable bowel syndrome.

We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.

肠易激综合征的循环miRNA的扰动检测使用复用高通量基因表达平台

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

科研

遗传学

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycERTAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3&#8217;UTRs by dual reporter plasmid transfection and dual luciferase assay.

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

一个人类神经干细胞系的三维文化，微小RNA的分析和推测的靶基因分化

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

发育生物学

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful biomarkers for clinical practice. Here, we present a protocol to extract, reverse transcribe and evaluate the expression levels of circulating miRNAs from plasma samples of patients with colorectal cancer (CRC). microRNAs (miRNAs) are a class of non-coding RNAs of 18-25 nucleotides in length that regulate the expression of target genes at the translational level and play an important role, including that of pro- and anti-angiogenic function, in the physiopathology of various organs. miRNAs are stable in biological fluids such as serum and plasma, which renders them ideal circulating biomarkers for cancer diagnosis, prognosis and treatment decision-making and monitoring. Circulating miRNA extraction was performed using a rapid and effective method that involves both organic and column-based methodologies. For miRNA retrotranscription, we used a multi-step procedure that considers polyadenylation at 3&#39; and the ligation of an adapter at 5&#39; of the mature miRNAs, followed by random miRNA pre-amplification. We selected a 24-miRNA custom panel to be tested by quantitative real-time polymerase chain reaction (qRT-PCR) and spotted the miRNA probes on array custom plates. We performed qRT-PCR plate runs on a real-time PCR System. Housekeeping miRNAs for normalization were selected using GeNorm software (v. 3.2). Data were analyzed using Expression suite software (v 1.1) and statistical analyses were performed. The method proved to be reliable and technically robust and could be useful to evaluate biomarker levels in liquid samples such as plasma and/or serum.

We present a protocol to evaluate the expression levels of circulating microRNA (miRNAs) in plasma samples from cancer patients. In particular, we used a commercially available kit for circulating miRNA extraction and reverse transcription. Finally, we analyzed a panel of 24 selected miRNAs using real-time pre-spotted probe custom plates.

转移性结直肠癌患者循环微 rna 自定义面板的检测

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful ...

癌症研究

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3&#8217; untranslated regions (3&#8217; UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

用于评估肿瘤细胞中的微RNA水平、功能和相关靶基因的体外协议

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

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 ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

在乳腺癌细胞雌激素调节小分子RNA的剖析

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

医学

An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying the binding partners of an RNA of interest remains of high importance to unveil the mechanisms behind many cellular processes. However, RNA molecules might interact transiently and dynamically with some RNA-binding proteins (RBPs), especially with non-canonical ones. Hence, improved methods to isolate and identify such RBPs are greatly needed.
To identify the protein partners of a known RNA sequence efficiently and quantitatively, we developed a method based on the pull-down and characterization of all interacting proteins, starting from cellular total protein extract. We optimized the protein pull-down using biotinylated RNA pre-loaded on streptavidin-coated beads. As a proof of concept, we employed a short RNA sequence known to bind the neurodegeneration-associated protein TDP-43 and a negative control of a different nucleotide composition but the same length. After blocking the beads with yeast tRNA, we loaded the biotinylated RNA sequences on the streptavidin beads and incubated them with the total protein extract from HEK 293T cells. After incubation and several washing steps to remove nonspecific binders, we eluted the interacting proteins with a high-salt solution, compatible with the most commonly used protein quantification reagents and with sample preparation for mass spectrometry. We quantified the enrichment of TDP-43 in the pull-down performed with the known RNA binder compared to the negative control by mass spectrometry. We used the same technique to verify the selective interactions of other proteins computationally predicted to be unique binders of our RNA of interest or of the control. Finally, we validated the protocol by western blot via the detection of TDP-43 with an appropriate antibody. 
This protocol will allow the study of the protein partners of an RNA of interest in near-to-physiological conditions, helping uncover unique and unpredicted protein-RNA interactions.

Here, we present an optimized in vitro method to uncover, quantify, and validate protein interactors of specific RNA sequences, using total protein extract from human cells, streptavidin beads coated with biotinylated RNA, and mass spectrometry analysis.

使用短生物素化RNA对RNA结合蛋白进行优化的定量下拉分析

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying ...

生物化学

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 ...

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 ...