这项工作得到了美国国立医学研究所 (NIH (P20GM103499).) 的 INBRE 赠款的支持, 并资助 CA555536 & CA154664 从国家癌症研究所到 P.H.H。这一方案得到部分支持, 从霍华德休斯医学院通过大学预科 & 本科生科学教育项目, 并由国家研究资源中心 (5 P20 RR016461) 赠款。和国立卫生研究院 (8 P20 GM103499)。

1. 培养和代谢标记
<ol><li>一次戳一个无菌1.5 毫升微离心管的中心, 用18口径的针头, 允许适当的曝气。接种500µL 的补充无菌7H9 汤与5µL 的m 耻从一夜成名的文化增长12。 注: 一公升7H9 汤的配方为: 4.7 克商业7H9 粉, 2 毫升甘油, 1 毫升吐温 80, 0.85 克氯化钠, 5 克白蛋白, 2 g 葡萄糖, H2O (使总体积到 1 L)。<ol>
<li>
(警告)按照标准的防护做法, 使用20µCi/mL 的 [32P] Na2HPO4来对区域性媒体进行尖峰。生长在37° c 和 1200 rpm 搅拌的细菌, 在一个9毫米厚的亚克力盾牌后面的振动筛孵化器。</li>
	</ol>
</li>
	<li>在 midlog 阶段 (外径 600 nm ~ 0.5), 将整个培养物转化为2毫升的螺旋帽管, 室温下的颗粒状放射性细菌离心为2分钟, 在 1万 x g。适当的废物容器。</li>
</ol>2. 总 rna 的制备
注: 根据制造商的协议, 使用商业 rna 提取试剂 (含硫氰酸胍、苯酚和氯仿) 从细菌小球中提取总 rna。
<ol><li>添加1毫升的商业 RNA 提取试剂和大约200µL 玻璃珠上的颗粒。在最大搅拌的情况下, 将管帽安全地盖在一个均质器内, 并将细菌扰乱2分钟 (在选定的仪器上设置编号五 (请参见材料表))。</li>
	<li>离心机样品在 1.2万 x g 为10分钟在4° c。将上清液转移到新的2毫升管, 并适当地丢弃珠子。十五年代用手用力加0.2 毫升氯仿和摇管. 离心机在 1.2万 x g 为15分钟在4° c。</li>
	<li>通过在45°上钓鱼, 将样品的水相收集到一个新的管子中。避免绘制任何界面或有机层。添加2µL 色沉淀和0.5 毫升100% 异丙醇。用手用力摇动管子5° c 1.2万 x g 的离心机10分钟。</li>
	<li>从管中取出上清液, 并适当地丢弃, 因为液体馏分可能含有放射性的痕迹。空气干燥颗粒为5分钟, 然后重在200µL 2X 生理盐水-柠檬酸钠 (SSC), 0.1% (瓦特/五) 十二烷基硫酸钠 (SDS) 决赛。 注:20 X SSC 的成份为3.0 米氯化钠, 0.3 米柠檬酸钠, pH 值7.0。</li>
</ol>3. 96 井印版的研制
注: tRNA 微阵列是手工打印的四十二70的 DNA 寡核苷酸互补的 3 ' 结束的每个m. 耻tRNA (终端共同国家评估排除) 使用八针机器人。DNA 寡核苷酸探针的序列和探针布局在 well-plate 和阵列被提供 (补充文件 1-板料1到3分别)。
<ol><li>设计 DNA 探针首先检索 tRNA 序列或 tRNA 编码基因从 tRNA 数据库, 如基因组 tRNA 数据库13。修剪保守3的最终评估, 如果编码。反向补充结果序列。基于前70核苷酸的顺序探针。</li>
	<li>重干 DNA 探针在水中。将探头浓度调整到100µM。在 96 well-plate, 准备120µL 50 µM 寡在 3X ssc, 0.01% (瓦特/v) sds 决赛 (稀释60µL 的股票探头在个人井与60µL 6X ssc, 0.01% (瓦特/v) sds。</li>
	<li>盖板与锡纸, 以防止蒸发或污染, 立即冻结, 并保持在-20 ° c, 直到需要。</li>
</ol>4. 阵列打印
<ol><li>在室温下解冻96井板 (大约需要20分钟)。</li>
	<li>用钻石笔将胺涂层玻璃贴上标签。将幻灯片放入索引单元。跟随网格安置并且做如下。<ol>
<li>小心地将复制器针脚浸入井中。</li>
		<li>轻轻地在玻璃上打印阵列, 使用最小的压力 (你会感觉到小的阻力)。继续打印, 不清洗机器人, 直到完成与块 A。</li>
	</ol>
</li>
	<li>当准备移动到下一个街区时, 按照下面描述的洗涤程序。<ol>
<li>在5% 漂白和轻轻摇动的倾角复制器。</li>
		<li>按复制器进入吸水纸。</li>
		<li>将复制体浸在蒸馏水中, 轻轻摇动, 然后按入纸。重复此步骤一次。</li>
		<li>在异丙醇上蘸复制器, 轻轻摇动, 然后按进纸张。</li>
		<li>将复制器在风扇上晾干大约二十年代, 然后再移动到96井板的下一个区块。继续打印所需的数量。</li>
	</ol>
</li>
	<li>允许幻灯片晾干, 然后用 254 nm 的 UV 交将寡与幻灯片交联。<ol>
<li>将能量级别设置为999990µJ/cm2。</li>
		<li>将幻灯片正面放在交的干净表面上。按 "开始"。</li>
	</ol>
</li>
	<li>在磁搅拌器上, 在室温和慢搅拌下, 用450毫升的堵塞溶液在一夜之间阻挡阵列。收集和重用阻塞解决方案最多四次。 注: 堵塞解决方案的配方是: 125 mm 磷酸盐缓冲液 (NaH2PO4/Na2HPO4) pH 7.2, 2.25% SDS (含/五), 0.5 毫米 EDTA, 0.5X SSC, 1% BSA (w/v)。</li>
	<li>在500毫升蒸馏水中, 在室温下5分钟洗涤2次幻灯片。用微阵列离心机将十年代的离心在室温下干燥。存储阵列在干燥和黑暗的地方长达6月。</li>
</ol>5. 阵列杂交
<ol><li>在杂交前, 用沸水冲洗滑块, 用离心法干燥 (如 4.6)。</li>
	<li>放置阵列内的杂交盒和负载标记 RNA 样本。</li>
	<li>运行自动杂交洗站的最大再现性。<ol>
<li>程序步骤如下。条件垫片为2分钟, 在75° c。在60° c 时引入探头。 变性探针和样品5分钟在90° c。杂交样品为3小时在60° c。<ol>
<li>在50° c 下两次冲洗幻灯片, 2X ssc, 0.1% (带 v) SDS, 十年代和二十年代举行. 在42° c 两次洗涤幻灯片与 0.1X ssc, 0.1% SDS, 流动十年代和持有二十年代. 在42° c 两次清洗幻灯片 0.1 X ssc, 流动十年代并且举行二十年代。</li>
		</ol>
</li>
	</ol>
</li>
	<li>离心干燥的幻灯片 (如 4.6)。</li>
</ol>6. 阵列量化
<ol><li>用薄薄的塑料薄膜包装幻灯片。在最敏感的设置上使用盖革计数器检查放射性信号的幻灯片。根据信号强度, 将幻灯片暴露在室温为10至90小时的曝光盒中的存储荧光屏上。 注意: 由于盖革计数器的灵敏度因仪器而异, 因此必须根据经验对曝光时间进行优化。</li>
	<li>使用 phosphorimager 扫描50µm 分辨率的幻灯片。使用微阵列探查器升级的免费图像 J 软件, 在每个探针点量化和背景减去放射性强度。</li>
	<li>使用电子表格组织和处理数据。使用条件格式工具生成热映射。例如代表每个探针信号作为分数 (用‰表示) 总探针信号。</li>
</ol>

<ol>
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M., Vinogradova, E., Salinas, T., Remacle, C., Mar&#233;chal-Drouard, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+evolution+and+expression+of+Chlamydomonas+reinhardtii+nucleus-encoded+transfer+RNA+genes.">On the evolution and expression of Chlamydomonas reinhardtii nucleus-encoded transfer RNA genes.</a> Genetics. 179 (1), 113-123 (2008).</li><li>Zheng, G., Qin, Y., Clark, W. C., Dai, Q., Yi, C., He, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+quantitative+high-throughput+tRNA+sequencing.">Efficient and quantitative high-throughput tRNA sequencing.</a> Nat Methods. 12 (9), 835-837 (2015).</li><li>Grelet, S., McShane, A., Hok, E., Tomberlin, J., Howe, P. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleiotropic+Roles+of+Non-Coding+RNAs+in+TGF-&#946;-Mediated+Epithelial-Mesenchymal+Transition+and+Their+Functions+in+Tumor+Progression.">Pleiotropic Roles of Non-Coding RNAs in TGF-&#946;-Mediated Epithelial-Mesenchymal Transition and Their Functions in Tumor Progression.</a> Cancers. 9 (7), (2017).</li><li>Belanger, A. E., Hatfull, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exponential-phase+glycogen+recycling+is+essential+for+growth+of+Mycobacterium+smegmatis.">Exponential-phase glycogen recycling is essential for growth of Mycobacterium smegmatis.</a> J Bacteriol. 181 (21), 6670-6678 (1999).</li><li>Chan, P. P., Lowe, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GtRNAdb:+A+database+of+transfer+RNA+genes+detected+in+genomic+sequence.">GtRNAdb: A database of transfer RNA genes detected in genomic sequence.</a> Nucl. Acids Res. 37 (Database issue), D93-D97 (2009).</li><li>Schmidt, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effect+of+Radioactive+Phosphorus+upon+a+Suspension+of+Escherichia+coli.">The Effect of Radioactive Phosphorus upon a Suspension of Escherichia coli.</a> J Bacteriol. 55 (5), 705-710 (1948).</li></ol>

没有利益冲突宣布。

β粒子的排放是已知的抑菌和杀菌剂14, 但是在测试范围内的辐射对细胞的适应性没有显著影响。闪烁计数表明, 放射性被纳入25% 的总细胞提取物, 随后, 1% 的纯化总 rna。
探测器的大小相同, 熔点和 GC 含量相同, 同时 macroarray 斑点、杂交和定量的统一协议允许直接从 spot 信号中测量 tRNA 水平。
现货是可重现和具体的。它的大动态范围和易于调整的门槛允许分析低丰富的物种, 如转运与聚, 只需延长阵列曝光时间9。在对所需设备进行初步投资后, 每个样品的运行费用, 包括消耗品, 平均每样15美元。
斑点适用于任何生物体, 其基因组可用于探针设计。在体外生长的模型生物体是新陈代谢标记的理想候选者。由于哺乳动物的基因组经常编码许多 isodecoders (转运, 共享相同的 anticodons, 但显示在他们的身体序列的细微差异) 探针需要部分退化, 以保存接近 tRNA 物种的同源杂交。最后, 与悬浮物中生长的生物体相比, 附着在哺乳动物细胞中的总 RNA 的数量要低得多, 例如m 耻, 所以文化需要大幅度地扩大。

细胞 tRNA 水平的波动根据条件, 如细胞类型, 环境, 和压力1,2,3。现场, 简化的观察 tRNA 的平台, 是一个原始的, 可靠的, 直接的技术, 允许快速和精确的量化的转移 RNA 水平的实验室培养的有机体。
转运具有显著稳定的二级和三级结构4。它们还显示许多后修改5。这些特征代表重要的结构和序列路障偏置, 或有时阻止使用标准的基准 RNA 量化技术6进行直接和可重现的 tRNA 分析。世界各地的研究小组被迫发展创造性的, 但往往是复杂的技术来评估细胞 tRNA 水平。其中的一些方法包括: (1) two-dimensional 电泳分离新陈代谢标记转运结合有条理的斑点分配的北印迹1;(2) 使用经过仔细标准化的放射性 DNA 探针的北印迹的个体量化7;(3) 蒜标记与菁荧光其次是微阵列分析3, 和 (4)体外剥离 tRNA 修饰使用重组 demodification 酶结合反转转录和随后高通量排序8。
斑点是两个标准和直接的方法的简单和原始的组合: (1) RNA 身体标记, 包括在生长的有机体, 放射性总 rna 和 (2) tRNA macroarrays, 系统和微型基因组工具优化 tRNA 隔离。
样品从活有机体被简化到定量平台。他们是直接分析后提取, 而不是进一步处理, 大大限制了偏见相比, 需要蒜放大或酶治疗技术。斑点是多才多艺的, 可以很容易地结合到 polysome 分馏, 以确定和量化 tRNA 亚群, 与翻译核糖体的物理联系。
此处介绍的协议针对分枝杆菌耻进行了优化, 并且还成功地用于在大肠杆菌9、酿酒酵母10、鼠标和人类细胞培养中对转运进行剖面分析11.

<table>
	<tbody>
		<tr>
			<td>Glass slide indexing system</td>
			<td>V&#38;P Scientific, Inc.</td>
			<td>VP 470</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slide arrayer replicator</td>
			<td>V&#38;P Scientific, Inc.</td>
			<td>VP 478</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well microplate indexing system</td>
			<td>V&#38;P Scientific, Inc.</td>
			<td>VP 472</td>
			<td></td>
		</tr>
		<tr>
			<td>Wash and blot station</td>
			<td>V&#38;P Scientific, Inc.</td>
			<td>VP 475</td>
			<td></td>
		</tr>
		<tr>
			<td>Replicator pin dryer</td>
			<td>V&#38;P Scientific, Inc.</td>
			<td>VP 474</td>
			<td></td>
		</tr>
		<tr>
			<td>Amine coated slides</td>
			<td>Molecular Devices</td>
			<td>K2615</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridizer / Automated hybridization and washing station</td>
			<td>Digilab</td>
			<td>Hyb10022</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker incubator</td>
			<td>Eppendorf Themomixer</td>
			<td>05-400-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw cap 2 ml tubes</td>
			<td>Thermo Scientific</td>
			<td>21-403-201</td>
			<td></td>
		</tr>
		<tr>
			<td>[32P] Na2HPO4</td>
			<td>Perkin Elmer</td>
			<td>NEX011001MC</td>
			<td></td>
		</tr>
		<tr>
			<td>Guanidinium thiocyanate-phenol-chloroform (Trizol)</td>
			<td>Invitrogen</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mm glass beads</td>
			<td>Research Products International Corp.</td>
			<td>9831</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini bead mill</td>
			<td>VWR</td>
			<td>25-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Storage phosphore screen</td>
			<td>Fujifilm</td>
			<td>BAS-IP SR 0813</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphorimager</td>
			<td>GE Healthcare</td>
			<td>Typhoon FLA7000</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA oligonucleotides</td>
			<td>IDT</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>UV crosslinker</td>
			<td>Spectroline</td>
			<td>Select series 254 nm</td>
			<td></td>
		</tr>
		<tr>
			<td>Microarray centrifuge</td>
			<td>Arrayit Corporation</td>
			<td>MHC110V</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycobacterium smegmatis</td>
			<td>ATCC</td>
			<td>700084</td>
			<td></td>
		</tr>
		<tr>
			<td>Single-use needles</td>
			<td>BD (Becton Dickinson)</td>
			<td>305185</td>
			<td></td>
		</tr>
		<tr>
			<td>2 ml centrifuge tube</td>
			<td>Fisherbrand</td>
			<td>14-666-317</td>
			<td></td>
		</tr>
		<tr>
			<td>Precipitant (GlucoBlue)</td>
			<td>Invitrogen</td>
			<td>AM9516</td>
			<td></td>
		</tr>
		<tr>
			<td>7H9 broth</td>
			<td>Difco</td>
			<td>271310</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Chemicals</td>
			<td>C298-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Chemicals</td>
			<td>A451-4</td>
			<td></td>
		</tr>
		<tr>
			<td>20 X SSC</td>
			<td>Lonza</td>
			<td>51205</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Fisher Bioreagents</td>
			<td>BP166-100</td>
			<td></td>
		</tr>
		<tr>
			<td>hybridisation cassette</td>
			<td>GE Healthcare</td>
			<td>63-0035-44</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Amresco</td>
			<td>M126-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin</td>
			<td>Spectrum Chemicals</td>
			<td>A3611</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Fisher Chemicals</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA (Ethylenediaminetetraacetic Acid)</td>
			<td>Fisher Bioreagents</td>
			<td>BP2482-500</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M Phosphate buffer pH 7.2</td>
			<td>Alfa Aesar</td>
			<td>AAJ63974AP</td>
			<td></td>
		</tr>
	</tbody>
</table>

metabolic labeling and profiling of transfer rnas using macroarrays

转移 rna (tRNA) 是丰富的短编码 RNA 种类, 通常是76到90核苷酸的长度。转运直接负责蛋白质的合成, 将密码的 mRNA 转化为氨基酸序列。长久以来, 转运被认为是缺乏监管功能的内部分子。然而, 越来越多的证据表明, 细胞 tRNA 水平在对应的变化条件, 如细胞类型, 环境, 和压力波动。tRNA 表达的波动直接影响基因的翻译, 有利于或抑制特定蛋白质的表达。最终理解蛋白质合成的动态需要开发能够提供高质量的 tRNA 剖面的方法。我们这里提出的方法被命名为 SPOt, 它代表了用于观察 tRNA 的流线型平台。斑点由三步开始以细胞培养的新陈代谢的标记与放射性磷酸盐, 其次胍硫氰酸盐-苯酚-氯仿提取放射性总 rna 和最后杂交在 in-house 打印macroarraystRNA 水平的估计, 量化的放射性强度在每个探针点。在这里提出的协议中, 我们在分枝杆菌耻mc2155 转运, 一个致病细菌经常被用作模型生物体来研究结核病。

三独立的生物复制表明, 在测试的生长条件下, 所有的m 耻转运都表示为高于背景级别 (图 1)。在这个特定的实验中, 观察到的每个探针的标准偏差包括 2% (Arg) 和 22% (胱氨酸 (配合力)), 中间值为 5% (图 1)。复制之间的假变化很大程度上受到阵列准备和杂交等因素的影响。高再现性是通过仔细和一致的阵列打印以及自动样品杂交实现的。tRNA 与 tRNA (TAC) 之间有5倍的差异, 分别是最高和最低的丰种。斑点显示 tRNA isoacceptors 的表达不均匀。Isoacceptors 是种, 持有相同的氨基酸, 但显示不同的子序列, 因此解码不同的密码在基因。例如, 所有丙氨酸 isoacceptors 在相似的水平被表达, 而最高和最低的精氨酸接受转运不同由3倍。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56898/56898fig1.jpg"> 图 1: 代表 tRNA macroarray 结果.(A)扫描细菌、鼠标和人类 macroarrays。m 耻数组只使用43探针, 而不是48只用于鼠标和人。因此, 细菌阵列显示40个空点 (每个阵列上的探针被复制八次), 与背景相混合。他们中的大多数都聚集在左上角。(B)在最佳增长条件下的m 耻中的 tRNA 配置文件的直方图和图表示形式。探针信号是平均的三独立实验 (包括细菌生长, 新陈代谢的标记, 提取和阵列杂交) 和被表达作为每一千价值总转运。三复制的标准偏差分别表示为误差线和橙色在直方图和图上的深浅。<a href="http://cloudflare.jove.com/files/ftp_upload/56898/56898fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays at JoVE.com

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

我们描述了一个原始的传输 RNA 分析平台名为 SPOt (简化的观察 tRNA 平台)。斑点同时测量所有转运在生物样品中的细胞水平, 仅三步, 并在不到24小时。

使用 Macroarrays 的转移 rna 的代谢标记和剖面分析

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for ...

metabolic-labeling-and-profiling-of-transfer-rnas-using-macroarrays

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

5.8K 次观看.  College of Charleston. 我们描述了一个原始的传输 RNA 分析平台名为 SPOt (简化的观察 tRNA 平台)。斑点同时测量所有转运在生物样品中的细胞水平, 仅三步, 并在不到24小时。

视频: Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

单细胞基因表达谱使用流式细胞仪和qPCR内部标准

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

科研

遗传学

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA&#183;RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

We report the protocols for the synthesis and purification of Peptide Nucleic Acid (PNA) oligomers incorporating modified residues. The biochemical and biophysical methods for the characterization of the recognition of RNA duplexes by the modified PNAs are described.

化学修饰肽核酸对单链 rna 双链 rna 的序列特异和选择性识别

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

使用斑马鱼作为体内模型系统分析 DNA 复制定时

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

线粒体 DNA 甲基化的精确检测方法

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To date, miRNAs have been implicated in a large number of biological and disease processes, which has signified the need for the reliable detection methods of miRNA transcripts. Here, we describe a detailed protocol for digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe-based miRNA detection, combined with protein immunostaining on mouse heart sections. First, we performed an in situ hybridization technique using the probe to identify miRNA-182 expression in heart sections from control and cardiac hypertrophy mice. Next, we performed immunostaining for cardiac Troponin T (cTnT) protein, on the same sections, to co-localize miRNA-182 with the cardiomyocyte cells. Using this protocol, we were able to detect miRNA-182 through an alkaline phosphatase based colorimetric assay, and cTnT through fluorescent staining. This protocol can be used to detect the expression of any miRNA of interest through DIG-labeled LNA probes, and relevant protein expression on mouse heart tissue sections.

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.

原位杂交法在小鼠心脏切片组织特异 miRNA 表达谱分析中的应用

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

酿酒酵母4-硫尿嘧啶代谢标记及新合成 mRNA 作为 RNA 聚合酶 II. 活性的一种代理

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

Cell Based Assays of SINEUP Non-coding RNAs That Can Specifically Enhance mRNA Translation

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. Here, we present a method to selectively up-regulate protein expression of desired genes in cultured cells by means of synthetic antisense non-coding RNAs known as SINEUPs. This positive control of gene expression is at the post-transcriptional level and exerted by an inverted short interspersed nuclear element (SINE) repeat at the 3&#697; end of SINEUPs that comprises its effector domain (ED). SINEUPs can specifically bind to any protein-coding mRNA of choice through its binding domain (BD), a region designed to complement the sequence within the 5&#697; untranslated region (5&#697; UTR) and around the start codon of the mRNA. Target-specific SINEUPs designed in this manner are transfected to cultured cells, and protein and RNA are extracted for downstream analyses, generally 24-48 h post-transfection. SINEUP-induced protein up-regulation is detected by Western-blot analysis and RNA expression is measured using real-time quantitative reverse transcription PCR. We have observed that BD design is critical for achieving optimum SINEUP activity and that testing different BD sizes and positions with respect to the start codon of the target mRNA is recommended. Therefore, we describe here a semi-automated high-throughput imaging method based on fluorescence detection that can be implemented to target mRNA fused with green fluorescent protein (GFP). SINEUPs specifically enhance translation within normal physiological range of the cell, without altering the target transcript level. This method has been successfully employed against a range of endogenous and exogenous targets, in a wide variety of human, mouse, and insect cell lines along with in vivo systems. Moreover, SINEUPs have been reported to increase antibody production and work as an RNA therapeutic against haploinsufficient genes. The versatile and modular nature of SINEUPs makes them a suitable tool for gene-specific translational control.

SINEUPs are synthetic antisense non-coding RNAs, which contain a binding domain (BD) and an effector domain (ED) and up-regulate translation of target mRNA. Here, we describe detection methods for SINEUPs in cultured cell lines, analysis of their translation-promoting activity by Western-blot and a semi-automated high throughput imaging system.

基于细胞的 sineup 非编码 rna 检测, 可专门增强 mrna 的翻译能力

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. ...

Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since 2007. These studies have confirmed that lncRNAs are altered in almost all diseases. However, studying the functional roles for lncRNAs in the context of disease remains difficult due to the lack of protein products, tissue-specific expression, low expression levels, complexities in splice forms, and lack of conservation among species. Given the species-specific expression, lncRNA studies are often restricted to human research contexts when studying disease processes. Since lncRNAs function at the molecular level, one way to dissect lncRNA biology is to either remove the lncRNA or overexpress the lncRNA and measure cellular effects. In this article, a written and visualized protocol to overexpress lncRNAs in vitro is presented. As a representative experiment, an lncRNA associated with inflammatory bowel disease, Interferon Gamma Antisense 1 (IFNG-AS1), is shown to be overexpressed in a Jurkat T-cell model. To accomplish this, the activating clustered regularly interspaced short palindromic repeats (CRISPR) technique is used to enable overexpression at the endogenous genomic loci. The activating CRISPR technique targets a set of transcription factors to the transcriptional start site of a gene, enabling a robust overexpression of multiple lncRNA splice forms. This procedure will be broken down into three steps, namely (i) guide RNA (gRNA) design and vector construction, (ii) virus generation and transduction, and (iii) colony screening for overexpression. For this representative experiment, a greater than 20-fold enhancement in IFNG-AS1 in Jurkat T cells was observed.

Traditional cDNA-based overexpression techniques have a limited applicability for the overexpression of long noncoding RNAs due to their multiple splice forms with potential functionality. This review reports a protocol using CRISPR technology to overexpress multiple splice variants of a long noncoding RNA.

使用基因激活 crispr 过度表达长非编码 rna

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since ...

Identification of Circular RNAs using RNA Sequencing

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein interactions, and regulation of parental gene transcription. In classical next generation RNA sequencing (RNA-seq), circRNAs are typically overlooked as a result of poly-A selection during construction of mRNA libraries, or are found at very low abundance, and are therefore difficult to isolate and detect. Here, a circRNA library construction protocol was optimized by comparing library preparation kits, pre-treatment options and various total RNA input amounts. Two commercially available whole transcriptome library preparation kits, with and without RNase R pre-treatment, and using variable amounts of total RNA input (1 to 4 &#181;g), were tested. Lastly, multiple tissue types; including liver, lung, lymph node, and pancreas; as well as multiple brain regions; including the cerebellum, inferior parietal lobe, middle temporal gyrus, occipital cortex, and superior frontal gyrus; were compared to evaluate circRNA abundance across tissue types. Analysis of the generated RNA-seq data using six different circRNA detection tools (find_circ, CIRI, Mapsplice, KNIFE, DCC, and CIRCexplorer) revealed that a stranded total RNA library preparation kit with RNase R pre-treatment and 4 &#181;g RNA input is the optimal method for identifying the highest relative number of circRNAs. Consistent with previous findings, the highest enrichment of circRNAs was observed in brain tissues compared to other tissue types.

Circular RNAs (circRNAs) are non-coding RNAs that may have roles in transcriptional regulation and mediating interactions between proteins. Following assessment of different parameters for construction of circRNA sequencing libraries, a protocol was compiled utilizing stranded total RNA library preparation with RNase R pre-treatment and is presented here.

使用RNA测序识别循环RNA

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein ...

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as plant microRNAs (miRNAs) carry a 2&#39;-O-methyl (2&#39;-OMe) modification at their 3&#39;&#160;terminal nucleotide. This modification adds another difficulty as it inhibits 3&#39;&#160;adapter ligation. We previously demonstrated that modified versions of the &#39;TruSeq (TS)&#39;&#160;protocol have less bias and an improved detection of 2&#39;-OMe RNAs. Here we describe in detail protocol &#39;TS5&#39;, which showed the best overall performance. TS5 can be followed either using homemade reagents or reagents from the TS kit, with equal performance.

We present a detailed small RNA library reparation protocol with less bias than standard methods and an increased sensitivity for 2&#39;-O-methyl RNAs. This protocol can be followed using homemade reagents to save cost or using kits for convenience.

改进小RNA-seq:减少偏差，更好地检测2'-O-甲基RNA

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as ...