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27,31的直接约束力。本报告详述了一个实验策略, 利用生物素化 miRNA 模拟到下拉靶 mRNA。生物素下拉可用于识别高灵敏度、低假阳性率35、43的 miRNA 靶。因此, 通过耦合序列筛选捕获的 RNA 池, 也可以利用该方法对 miRNA 目标进行识别。但是, 此方法44有几个限制。外源性 miRNA 的过度表达可以改变细胞的转录网络, 从而导致 mRNA 的变化, 而不是由于 miRNA 调节。这些可以通过使用极低的模拟器来克服, 以免扰乱内生 miRNA 调节。另外, 当 miRNAs 被外部表达以避免非特定绑定时, 需要适当的控件。仔细的洗涤和堵塞步骤可以有效地减少背景噪声, 增加目标 miRNA 的特异性。很少有研究表明, 对 miRNA 3 ' 或 5 ' 末端的修改是不能容忍的45, 然而, 一些研究已经有效地使用生物素标记的和平号模拟器作为一个有效的工具来探索 mRNA 目标。
这种方法的另一个优点是利用低噪声的 (锁定核酸) miRNA 模拟。低噪声的核酸修饰允许形成稳定的寡核苷酸复式与高亲和力46,47。此外, 定制的生物素化低噪声 miRNA 模拟包括三 RNA 链。一个 3 '-生物素化 miRNA 链与序列的 miRBase 注释和乘客链互补的 miRNA, 这是分裂的两个低噪声的改进 RNA 链47。RISC 仅包含 miRNA 链, 而两条乘客链迅速退化, 从而增强目标特异性。为了提高我们的发现的信心, 我们还包括了一个验证的目标 p53 的 miR-125b 作为一个积极的控制和β肌动蛋白, 它没有一个 miR-125b 绑定网站在其 mRNA 作为一个负控制。使用此方法得到的结果与这些控制 (图 3) 显示了目标 mRNA 识别的特异性。虽然低噪声放大 miRNA 模仿稳定, 并大大有利于沃森-克里克基地配对, 应该指出, 使用低噪声放大 miRNA 模仿生物素标签将消除不假阳性和阴性。此外, 他们可能会导致确认的计算预测, 可能不是生物学上的相关。另一方面, 如果这种方法与测序相结合以确定 mRNA 目标, 可能会产生假阴性, 因为低噪声放大模拟倾向于规范基配对, 这可能排除与本机 miRNAs 发生的非规范基配对交互。
这里提出的协议是由迩和艾尔改编的, 有几个修改35。为了进一步减少背景噪声, 我们结合了广泛的洗涤步骤, 修改了转染效率的参数, 缓冲准备和孵化, 下拉条件和 PCR 数据的采集和分析。此外, 我们使用两个引物集放大的目标 mrna 后, 下拉, 一组旨在放大一个区域的目标 mrna 和第二套底漆, 以放大一个区域内的 3 ' UTR 的目标 mrna。两套引物的利用提高了靶 mRNA 富集的特异性。
应当指出的是, 这种方法的局限性是由于非特定目标而产生的基本噪音水平。进一步的修改, 如堵塞, 洗涤和孵化条件的改善可能会提供更高的目标特异性和敏感性。总之, 本报告所描述的化验是一种快速可靠的方法, 可用于验证 miRNAs 的 mRNA 目标使用 PCR 策略。

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&#8217;s modified Eagle&#8217;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%)&#160;</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&#160;</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 (MgCl2) 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&#160;</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36,37,38。我们采用这种方法将 PARP-1 mRNA 作为细胞 miRNA miR-125b 的靶向。在调节神经元功能39、40的脑组织中检测到最高的 miR-125b 表达水平。为了确定神经元细胞中 miR-125b 的靶点, 我们最近报告说, miR-125b 对 PARP-1 mRNA41的 3 ' UTR 的 PARP-1 表达有消极的调节。
miRNA 的下拉: mRNA 复合物
我们利用 HEK-293T 细胞研究 miR-125b 和 PARP-1 mRNA 之间的相互作用, 因为这些细胞广泛应用于细胞、生物化学和分子生物学研究。图 1描述了用于 mRNA 目标的协议示意图。首先, HEK-293T 细胞 (4 x 105–5 x 105细胞每井) 被播种隔夜在6个井组织培养板材和孵化在37°c 与 5% CO2为 18–24 h。随后, 采用脂质体转染法, 将 75 picomoles 3 '-生物素化 miR-125b 模拟器和 3 '-生物素化炒控器转化为细胞。转染的细胞在37°c 和 5% CO2被孵化了另外 36 h 和收获。然后用冻融裂解法制备转染细胞的细胞裂解物。新制备的细胞裂解物在室温下, 在工作台顶 nutating 搅拌机上用被阻挡的链亲和素涂层磁性珠子孵化。在此之后, 用刚准备好的冰冷下拉式洗涤缓冲器在磁选机上加工和洗涤珠子。最后, 在100µL 核酸酶自由水中悬浮珠, 进一步分析。
下拉 miRNA 中靶 mrna 的检测: mRNA 复合物
为了从下拉合剂中检测 miR-125b 的靶 mRNA, 我们采用了 qPCR 法 (图 1)。我们设计了在 ORF 内的向前和反向引物 (FP 和 RP) 放大 PARP-1 mRNA (图 2, 表 5b)。我们还设计了用于检测 p53 mRNA (miR-125b42的已知目标) 的引物集, 作为一种阳性对照, 包括将肌动蛋白 mRNA 放大为非特定阴性对照的引物。此外, 为了进一步确定放大的特异性, 我们设计了用于检测 PARP-1、p53 和肌动蛋白 3 ' UTR 区的底漆。我们使用协议中描述的方法执行 qPCR (6 节)。
图 3显示了基于 qPCR 的目标 mRNA 相对于被纯化珠的炒控制的放大。与生物素化 miR-125b 模拟相比, PARP-1 mRNA 的含量明显高于被炒的对照组。如预期, 在这些样品中观察到阳性对照 p53 mrna 的较高 mrna 水平和负控制肌动蛋白 mrna 的最小扩增。与负控制肌动蛋白 (图 3B) 相比, 使用引物靶向 3 ' UTR PARP-1 和 p53 的底漆获得了类似的扩增水平。qPCR 的结果 (图 3A) 和 3 ' UTR 区域 (图 3B) 的 PARP-1 mrna 和 p53 mrna 显示了一定程度的变异性。一些因素, 包括绑定亲和性, miRNA 绑定站点的数量, 以及引物序列依赖放大的差异可能会导致在图 3中放大的可变水平。然而, 这些数据有力地支持验证细胞 miRNAs mRNA 目标的方法的可行性和特异性。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57786/57786fig1.jpg"> 图 1: 使用生物素化 miRNA 模拟器的目标捕获检测协议的示意图表示.<a href="/files/ftp_upload/57786/57786fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57786/57786fig2.jpg"> 图 2: 用于放大 mRNA 目标的底漆设计.用于检测 miR-125b 靶 mRNA 的引物的示意图。一组引物 (套 I) 是设计在 ORF 地区的成绩单和另一套引物 (设置 II) 是设计在 3 ' UTR 地区的目标基因。FP 和 RP 表示每个集合的正向和反向引物。<a href="/files/ftp_upload/57786/57786fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57786/57786fig3.jpg"> 图 3: miR-125b 目标识别 qPCR.(A)使用入门组 I 的 mRNA 水平, 放大目标 mRNA 的 ORF 区域。与肌动蛋白 mrna (阴性对照) 相比, 观察到 PARP-1 和 p53 mrna (阳性对照) 的表达水平较高。(B)利用引物集对目标 mRNA 的 3 ' UTR 区域进行扩增。没有在无模板控件 (NTC) 中观察到放大。在 triplicates 中进行了所有的反应, 并利用基于 2ΔCt方法的数据分析软件对数据进行了分析。误差线表示平均值的标准误差。<a href="/files/ftp_upload/57786/57786fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>组件</td>
			<td>股票</td>
			<td>20 ul 反应的最终体积</td>
		</tr>
<tr>
<td>模板 (RNA)</td>
			<td>50 ng/µL</td>
			<td>1.0 ul</td>
		</tr>
<tr>
<td>反应缓冲器</td>
			<td>5x</td>
			<td>4.0 ul</td>
		</tr>
<tr>
<td>寡聚 dT 底漆</td>
			<td>主库存</td>
			<td>2 ul</td>
		</tr>
<tr>
<td>逆转录</td>
			<td>主库存</td>
			<td>1 ul</td>
		</tr>
<tr>
<td>核酸酶无水蒸馏水</td>
			<td>-</td>
			<td>12 ul</td>
		</tr>
<tr>
<td>总反应量</td>
			<td></td>
			<td>20 ul</td>
		</tr>
</tbody></table>表 1: cDNA 合成反应混合物。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>105°c = 三十年代</td>
		</tr>
<tr>
<td>42°c = 60 分钟</td>
		</tr>
<tr>
<td>95°c = 5 分钟</td>
		</tr>
<tr>
<td>10°c = 举行</td>
		</tr>
</tbody></table>表 2: cDNA 合成热循环条件。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>组件</td>
			<td>股票</td>
			<td>10 ul 反应的最终体积</td>
		</tr>
<tr>
<td>cDNA 产品</td>
			<td>-</td>
			<td>1.0 ul</td>
		</tr>
<tr>
<td>iTaq 通用 SYBR 混合</td>
			<td>2x</td>
			<td>5.0 ul</td>
		</tr>
<tr>
<td>目标 (ORF/3 ' UTR) 平安险</td>
			<td>10微米</td>
			<td>0.5 ul</td>
		</tr>
<tr>
<td>目标 (ORF/3 ' UTR) R.P。</td>
			<td>10微米</td>
			<td>0.5 ul</td>
		</tr>
<tr>
<td>核酸酶无水蒸馏水</td>
			<td>-</td>
			<td>3 ul</td>
		</tr>
<tr>
<td>总反应量</td>
			<td></td>
			<td>10 ul</td>
		</tr>
</tbody></table>表 3: qPCR 反应混合物。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>95°c = 10 分钟</td>
		</tr>
<tr>
<td>30周期:</td>
		</tr>
<tr>
<td>95°c = 十年代</td>
		</tr>
<tr>
<td>56°c = 三十年代</td>
		</tr>
<tr>
<td>72°c = 三十年代</td>
		</tr>
<tr>
<td>熔体曲线分析</td>
		</tr>
<tr>
<td>65°c = 三十一年代</td>
		</tr>
<tr>
<td>线性坡道率 = 0.5 °c/秒</td>
		</tr>
<tr>
<td>承购 = 0.5 °c 间隔</td>
		</tr>
</tbody></table>表 4: qPCR 热循环条件。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="3">(A): 生物素化寡核苷酸</td>
		</tr>
<tr>
<td>Biotinlyated 寡核苷酸</td>
			<td>股票</td>
			<td>序列 (5 ' 到 3 ')</td>
		</tr>
<tr>
<td>has-miR-125b</td>
			<td>25微米</td>
			<td>UCCCUGAGACCCUAACUUGUGA-生物素</td>
		</tr>
<tr>
<td>炒控</td>
			<td>25微米</td>
			<td>GAUGGCAUUCGAUCAGUUCUA-生物素</td>
		</tr>
<tr>
<td>(B) 底漆</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>目标底漆 (ORF)</td>
			<td>股票</td>
			<td>序列 (5 ' 到 3 ')</td>
		</tr>
<tr>
<td>PARP-1 (FP)</td>
			<td>100微米</td>
			<td>GAGGTGGATGGGTTCTCTGA</td>
		</tr>
<tr>
<td>PARP-1 (RP)</td>
			<td>100微米</td>
			<td>ACACCCCTTGCACGTACTTC</td>
		</tr>
<tr>
<td>p53 (FP)</td>
			<td>100微米</td>
			<td>TGTGACTTGCACGTACTCCC</td>
		</tr>
<tr>
<td>p53 (RP)</td>
			<td>100微米</td>
			<td>ACCATCGCTATCTGAGCAGC</td>
		</tr>
<tr>
<td>肌动蛋白 (FP)</td>
			<td>100微米</td>
			<td>GCTCGTCGTCGACAACGGCTC</td>
		</tr>
<tr>
<td>肌动蛋白 (RP)</td>
			<td>100微米</td>
			<td>CAAACATGATCTGGGTCATCTT</td>
		</tr>
<tr>
<td>(C) 底漆</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>目标底漆 (3 ' UTR)</td>
			<td>股票</td>
			<td>序列 (5 ' 到 3 ')</td>
		</tr>
<tr>
<td>PARP-1 (FP)</td>
			<td>100微米</td>
			<td>ATTGGGAGAGGTAGCCGAGT</td>
		</tr>
<tr>
<td>PARP-1 (RP)</td>
			<td>100微米</td>
			<td>CTACCCATCAGCAACTTAGCG</td>
		</tr>
<tr>
<td>p53 (FP)</td>
			<td>100微米</td>
			<td>GGCCCATATCTGTGAAATGC</td>
		</tr>
<tr>
<td>p53 (RP)</td>
			<td>100微米</td>
			<td>CTGCAGGAAGGCAGGTTTT</td>
		</tr>
</tbody></table>表 5: 寡核苷酸的名称和序列。

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; ...

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

Nanyang Technological University

Research

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

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

肽衍生的方法在运输跨越植物细胞的基因和蛋白质和细胞器障碍

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

暴露在生理条件下的氧气在人体细胞中帽结合蛋白质分析

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

激光 Microirradiation 研究体内细胞对简单复杂 DNA 损伤的反应

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can potentially increase risk of sudden unexpected death in epilepsy (SUDEP). Electroencephalography (EEG) and electrocardiography (ECG) are widely used clinical diagnostic tools to monitor for abnormal brain and cardiac rhythms in patients. Here, a technique to simultaneously record video, EEG, and ECG in mice to measure behavior, brain, and cardiac activities, respectively, is described. The technique described herein utilizes a tethered (i.e., wired) recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Compared to wireless telemetry recording systems, the tethered arrangement possesses several technical advantages such as a greater possible number of channels for recording EEG or other biopotentials; lower electrode costs; and greater frequency bandwidth (i.e., sampling rate) of recordings. The basics of this technique can also be easily modified to accommodate recording other biosignals, such as electromyography (EMG) or plethysmography for assessment of muscle and respiratory activity, respectively. In addition to describing how to perform the EEG-ECG recordings, we also detail methods to quantify the resulting data for seizures, EEG spectral power, cardiac function, and heart rate variability, which we demonstrate in an example experiment using a mouse with epilepsy due to Kcna1 gene deletion. Video-EEG-ECG monitoring in mouse models of epilepsy or other neurological disease provides a powerful tool to identify dysfunction at the level of the brain, heart, or brain-heart interactions.

Here, we present a protocol to record brain and heart bio signals in mice using simultaneous video, electroencephalography (EEG), and electrocardiography (ECG). We also describe methods to analyze the resulting EEG-ECG recordings for seizures, EEG spectral power, cardiac function, and heart rate variability.

同步视频脑电图-心电图监测对小鼠癫痫模型 Neurocardiac 功能障碍的鉴别

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can ...

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding RNA&#39;s family. Although their biological functions remain largely unknown, the number of these lncRNAs has steadily increased and it is now estimated that humans may have more than 10,000 such transcripts. Some of these are known to be involved in important regulatory pathways of gene expression which take place at the transcriptional level, but also at different steps of RNA co- and post-transcriptional maturation. In the latter cases, RNAs that are targeted by the lncRNA have to be identified. That&#39;s the reason why it is useful to develop a method enabling the identification of RNAs associated directly or indirectly with a lncRNA of interest.
This protocol, which was inspired by previously published protocols allowing the isolation of a lncRNA together with its associated chromatin sequences, was adapted to permit the isolation of associated RNAs. We determined that two steps are critical for the efficiency of this protocol. The first is the design of specific anti-sense DNA oligonucleotide probes able to hybridize to the lncRNA of interest. To this end, the lncRNA secondary structure was predicted by bioinformatics and anti-sense oligonucleotide probes were designed with a strong affinity for regions that display a low probability of internal base pairing. The second crucial step of the procedure relies on the fixative conditions of the tissue or cultured cells that have to preserve the network between all molecular partners. Coupled with high throughput RNA sequencing, this RNA pull-down protocol can provide the whole RNA interactome of a lncRNA of interest.

This RNA pull-down method allows identifying the RNA targets of a long non-coding RNA (lncRNA). Based on the hybridization of home-made, designed anti-sense DNA oligonucleotide probes specific to this lncRNA in an appropriately fixed tissue or cell line, it efficiently allows the capture of all RNA targets of the lncRNA.

rna 下拉法识别长非编码 rna 的 rna 靶点

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding ...

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

We describe a method for generating glmS-based conditional knockdown mutants in Plasmodium falciparum using CRISPR/Cas9 genome editing.

CRISPR/Cas9 基因编辑制作人类疟疾寄生虫的条件突变体

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

RNA原位杂交法定量分析小鼠脑切片中的替代前 mRNA 拼接方法

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

利用体外和细胞内形状研究小分子诱导的预 mrna 结构变化

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

使用哺乳动物细胞中的微球菌核酸酶进行染色质免疫沉淀测定

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...