Name: determining genome-wide transcript decay rates in proliferating and quiescent human fibroblasts
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文森特卡塞尔是丽塔. 艾伦基金会 (http://www.ritaallenfoundation.org) 的弥尔顿学者。这项工作的经费来自国家普通医学科学研究所授予的 P50 GM071508 (私家侦探大卫博特斯坦), PhRMA 基金会赠款 2007RSGl9572, 国家科学基金会授予 OCI-1047879 8月大卫,国立普通医学科学院 R01 GM081686, 国立普通医学科学院 R01 GM0866465, 伊莱 & #38; 伊蒂丝再生医学和 #38 的广泛中心; 干细胞研究, 虹膜康托妇女健康中心/加州大学洛杉矶分校 CTSI 国立卫生研究院格兰特 UL1TR000124 和白血病淋巴瘤协会这份出版物中报告的研究得到了国家卫生研究院国家癌症研究所的 P50CA092131。资助者在研究设计、数据收集和分析、决定出版或准备原稿方面没有作用。是 Eli 和 #38 的成员; 伊蒂丝广泛的再生医学和 #38 中心; 干细胞研究, 加州大学洛杉矶分校分子生物学研究所, 加州大学洛杉矶分校生物信息学部际计划。

所有的实验都是由普林斯顿大学和加州大学洛杉矶分校的机构评审委员会批准的。
1. 制备增殖和接触抑制成纤维细胞 ActD 时间课程
注意: 本协议使用 timecourse 四 timepoints。三生物独立样品可以收集每 timepoint 通过收集不同的组织培养板在一个实验, 或者实验可以重复多次与细胞的不同的文化。在我们的经验, 一个10厘米 (直径) 组织培养皿 (一盘) 提供足够的 RNA 进行分析。如果需要, 可以为每个样本汇集多个组织培养皿, 以增加每个 timepoint 的细胞数量。此外, 同样的实验可以进行与孤立的成纤维细胞从不同的人真正独立的生物复制。
<ol>
	<li>建立细胞增殖和静止培养的转录衰变分析。对于增殖细胞, 每三天分裂细胞, 而接触抑制细胞则成为接触抑制。
	<ol>
		<li>对于接触抑制细胞, 每2天改变培养基7天。在接触抑制的成纤维细胞准备收集前2天分裂增殖细胞。图 1中显示了一个单元格区域性方案的示例。本节中的其余步骤提供了用于建立和拆分单元格的详细协议。</li>
	</ol>
	</li>
	<li>根据已建立的协议, 从新生儿皮肤中分离原发真皮成纤维细胞13。传代成纤维细胞, 以确保均匀的人口。 注: 成纤维细胞可以冷冻和解冻, 如果需要的话。</li>
	<li>如果需要, 解冻或 replate 成纤维细胞。在37° c 和 5% CO2的组织培养箱中, 在无菌条件下 DMEM 10% 血清培养成纤维细胞。使用已经生长了少于10通道的成纤维细胞。成纤维细胞应该和 #60; 80% 汇合在分裂细胞的天使增殖和淡静人口。</li>
	<li>将组织培养皿分成多个板, prewarm PBS、胰蛋白酶、培养基与血清在37° c 水浴20分钟。</li>
	<li>抽吸或使用吸管从每个组织培养板中取出培养基, 细胞 replated。</li>
	<li>吸管暖 PBS 到每个板块 (10 毫升的解决方案为150毫米板, 5 毫升的解决方案为100毫米板, 2 毫升为一个6井板, 1 毫升为一个井的12井板)。</li>
	<li>从每个组织培养板中抽出或吸管去除 PBS。</li>
	<li>轻轻地吸管1x 胰蛋白酶-EDTA 到每个板块 (8 毫升的解决方案为150毫米板, 3 毫升的解决方案为100毫米板, 2 毫升的一个井的6井板, 1 毫升的井的12井板)。 注: 用无菌10x 胰蛋白酶-edta 在无菌 PBS 中进行1x 胰蛋白酶-edta 的稀释。最后的1x 溶液应为0.05% 胰蛋白酶和 0.02% EDTA。</li>
	<li>用细胞孵育胰蛋白酶5分钟。</li>
	<li>轻轻拍打盘子, 将细胞从盘子里取出。</li>
	<li>吸管重细胞进入单个细胞悬浮。</li>
	<li>将胰蛋白酶溶液中的细胞转移到含有10% 血清的 DMEM 的锥形管中 (8 毫升的溶液用于 150 mm 板, 100 mm 板的3毫升溶液, 2 个井板的一个。, 1 毫升的12井板的井)。</li>
	<li>将细胞在4° c 下旋转5分钟, 在 1000 x g处去除胰蛋白酶。</li>
	<li>重细胞在适当的培养基量和计数与 hemacytometer, 以确定细胞浓度。</li>
	<li>种子 5 x 105细胞每100毫米组织培养板增殖和接触抑制细胞。</li>
	<li>使用此方法, 建立分析所需的增殖和接触抑制样本 (图 1)。</li>
</ol>
2. ActD 时间课程
<ol>
	<li>重 ActD 的浓度为1毫克/毫升 (1 毫克的 ActD, 使用950µL 无菌 PBS 和50µL 无菌的亚砜)。 注: 冷冻库存溶液的 ActD 应稳定在-20 ° c 的一个月。稀溶液应丢弃, 而不应储存以供进一步使用。</li>
	<li>为0、120、240和 480 min timepoints (图 2) 设置的所有生物复制细胞培养板添加 ActD 到适当的 prewarmed 培养基的体积。添加 ActD 浓度为15µg 每毫升培养基。混合好, 抽离旧培养基, 并在细胞中添加含有 ActD 的培养基。
	<ol>
		<li>对于1毫克/毫升的股票, 增加15µL ActD 为每毫升培养基,例如, 为10毫升的介质为100毫米板, 添加150µL ActD。</li>
	</ol>
	</li>
	<li>收集零 timepoint 样品, 轻轻地从 ActD 培养基中抽出, 吸管 prewarmed PBS 到细胞上。吸管10毫升的解决方案为150毫米板, 5 毫升的解决方案为100毫米板, 2 毫升的一个井的6井板, 1 毫升为一个井的12井板。</li>
	<li>轻轻地吸气或吸管 PBS。 注意: 所有涉及 RNA 隔离的步骤都应使用无核糖核酸的水、无核糖核酸的离心管和无核糖核酸的管。使用 RNA 时, 应佩戴手套, 并经常更换。</li>
	<li>添加苯酚-胍异硫氰酸酯溶液 (1 mL/10-cm 板, 4 x 105到 4 x 107细胞) 直接到组织培养板。 注: 苯酚-胍基异硫氰酸酯溶液是一种单相溶液, 能保持 rna 质量, 同时溶细胞, 分离 rna、DNA 和蛋白质。 注意: 苯酚和异硫氰酸胍具有腐蚀性。使用适当的保护。</li>
	<li>吸管的苯酚-胍异硫氰酸酯溶液-细胞混合物上下几次, 使一个均匀的混合物。 注: 苯酚-胍异硫氰酸酯溶液可在4° c 过夜或在-20 ° c 以下贮存一年。</li>
	<li>通过使用步骤 2.3-2.6 中描述的方法, 在适当的时间段后收集每个时间点。</li>
</ol>
3. 苯酚-胍基异硫氰酸酯溶液中总 RNA 的分离
<ol>
	<li>在室温下孵育样品5分钟, 以确保 RNA 蛋白复合物完全溶解。 注: 除非另行说明, 苯酚-胍异硫氰酸酯溶液协议的步骤可以在室温下进行。</li>
	<li>引入 spike-in 控制誊本, 使用方法可以检测到。</li></ol>在每个样品中以已知的数量和浓度引入这些控制。 注意: 外部 rna 控制联合体 (ERCC) spike-in 控制组合14被专门设计为用于 rna Seq 的 spike-in 控制。它也可用于北方杂交或 real-time qPCR。还提供了专门为微阵列技术设计的 Spike-in 控件 (请参见材料表)。
	<li>将苯酚-胍异硫氰酸酯溶液混合物转化为2.0 毫升的离心管和吸管。将0.2 毫升氯仿添加到苯酚-胍异硫氰酸酯溶液混合物中, 用于每1毫升苯酚-胍基异硫氰酸酯溶液 (例如, 将0.3 毫升氯仿添加到1.5 毫升异硫氰酸胍溶液混合物)。</li>
	<li>十五年代大力摇动离心管, 然后在室温下孵育氯仿-苯酚-胍异硫氰酸酯混合物3分钟。</li>
	<li>在 1.2万 x g上旋转样品20分钟, 在4° c。 注: 在初始旋转之后, 样品应该分成3层--底部是一个红色的有机层, 中间是白色/粉红色相间的, 上面是一个透明的水层 (图 3)。</li>
	<li>使用微将水层转移到新的2.0 毫升离心管。 注: 在这个过程中, 注意不要干扰界面。留下一些水的部分是可以的。最好留下一些含水率的部分, 而不包括某些相间的层, 包括界面层会污染 RNA 与 DNA。该水层将约为初始体积的一半, 所以约0.9 毫升, 如果苯酚-胍基异硫氰酸酯溶液/氯仿混合物为1.8 毫升。</li>
	<li>放弃相间和有机组分。</li>
	<li>在水中加入等量的 2-丙醇, 并将该管倒转10次。增加多达1µL 20 毫克/毫升水溶液的糖原每20µL 样品, 特别是如果产量预期是低的, 因为少于 106细胞收集。 注: 这将导致一个致密, 固体颗粒, 易于监测后, 自旋步骤, 随后。</li>
	<li>室温下孵育样品10分钟。</li>
	<li>在 1.2万 x g上旋转样品20分钟, 在4° c。确保, 在这个旋转的末尾, RNA 已经沉淀形成一个白色的小球在底部的管。</li>
	<li>用吸管取出并丢弃上清液, 注意不要干扰白色 RNA 颗粒。 注意: 手上握着的松吸管的尖端可以用来除去过量的乙醇。如果 RNA 颗粒扰 , 但没有被丢弃 , 研究者可以重复旋转 , 并再次去除上清。避免丢弃小球, 因为这将要求调查员重复整个过程。</li>
	<li>准备75% 乙醇和25% 无核酸水的溶液。每1毫升的苯酚-胍异硫氰酸酯溶液试剂加入1毫升的75% 乙醇, 清洗颗粒。</li>
	<li>旋转样品在 1.2万 x g为10分钟在4° c。</li>
	<li>除去大部分上清液后, 使用吸管去除尽可能多的乙醇, 同时还要注意不要扰乱颗粒。 注: RNA 颗粒在这一步是较不紧凑的, 并倾向于移动。在这一点上处理小球时要格外小心, 以避免失去 RNA。</li>
	<li>旋转样品 1.2万 x g在室温下2分钟, 以颗粒所有过量的乙醇。</li>
	<li>从管中取出所有过量的乙醇, 使用微, 注意不要扰乱颗粒。</li>
	<li>让 RNA 颗粒空气干燥5分钟。</li>
	<li>将50µL 的核酸水添加到 RNA 颗粒中, 并在无核酸水中重颗粒。</li>
	<li>用1µL dnasei (2 单位/µL) 来处理样品, 除去 DNA, 然后钝化 dnasei 和阳离子 (请参阅材料表)。</li>
	<li>在-80 ° c 的2.0 毫升离心管中贮存 RNA 样品。</li>

4. 成绩单丰度分析
<ol>
	<li>用分光光度计对 rna 进行量化并检查 rna 的纯度。 注: 260 nm 至 280 nm 的吸光度比应为 RNA 的大约2.0。</li>
	<li>分析 RNA 与1% 琼脂糖凝胶或等效技术, 以可视化程度的退化。 注意: 表示18S 和 28S rRNA 的两个清晰的频带应该清晰可见 (图 4)。如果这些波段不可见, RNA 可能会退化。讨论中提供了 RNA 降解的故障排除技巧。</li>
	<li>使用微阵列15、rna Seq16、real-time qPCR17或北印迹18, 与 spiked-in 内部标准相比, 在收集的 rna 等分中监控成绩单丰度。
	<ol>
		<li>与已知丰度的 spiked-in 控制相比, 在每个时间点确定每份成绩单的丰度。 注: 对于微阵列, 这些值将是荧光强度。对于北印迹, x 射线胶片上的波段可以量化。对于 real-time qPCR, 可以通过与已知标准与 2-σσCT方法19的比较来确定成绩单的丰度。对于 RNA-Seq, 规范化的值可以确定为 FPKM 或碎片每 kilobase 的外显子每百万读取映射。对于型特定的衰变率, 可以使用型的方法 (如 DEXSeq20) 分析 RNA Seq 数据。型特异性衰变率最好用 RNA-Seq 来确定。如果要用微阵列数据来确定这些数据, 那么就必须以探针为基础, 而不是逐个基因地分析。分析人员在从数量有限的探头中解释信息时必须小心。例如, 可能有5或更少的探测器提供有关特定型的信息。在这种情况下, 分析人员将需要确定这些探测器的行为是否足够一致, 以便结果可靠。</li>
	</ol>
	</li>
	<li>对收集的样本中的孤立 RNA17执行 real-time qPCR, 以分析迅速腐烂的基因, 如 c-myc 和 GAPDH 等缓慢衰减的基因, 以获得准确检测到转录衰变率的信心。 注: 为进一步确认, 可以开发和使用型特定的 real-time qPCR 探针, 以监测特定异构体的丰度, 时间21。</li>
</ol>
5。衰变率常数测定
<ol>
	<li>对于每个 timepoint, 日志转换每个成绩单的丰度值 (特定于基因或型)。 注意: 对数转换值将将指数衰减曲线转换为可与线性衰减模型22相匹配的行。</li>
	<li>将每个成绩单 (特定于基因或型) 的表达式配置为线性衰减模型。此类模型的公式为 f (t) = c-r t。在这个等式中, C 是一个常量, 代表了成绩单的起始量, r 是递减率, t 是自实验开始以来的时间。从这个等式, 确定衰变率 r * ln (10) (参见参考11)。 注: maSigPro 软件包是一种回归的方法, 旨在检测时间过程数据23中显著的基因表达谱差异。maSigPro 的示例代码和结果可在: https://www.bioconductor.org/packages/release/bioc/vignettes/maSigPro/inst/doc/maSigProUsersGuide.pdf。</li>
	<li>过滤掉无法适应对数线性衰减模型的基因。这可以通过方差分析 F 测试来完成。 注: f 检验基于 f 统计, 定义为样本间的变化除以样本内的变化。通过应用线性升压 Benjamini 和霍赫贝格错误发现过程24来更正多个比较的 p 值。一个 q 值大于0.05 与 f-测试可以被用来作为一个截止基因, 不能适应对数线性衰减模型。</li>
	<li>在分类细胞周期条件和时间 (假发现率, 罗斯福和 #60; 0.05) 中, 进行方差分析。</li>
</ol>

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作者没有竞争利益透露。

静止可由外部信号诱发, 包括 mitogens 或血清的提取、细胞黏附缺乏和接触抑制。接触抑制是诱发静止的多种可能的方法之一, 是一种高度进化的保守过程, 细胞在反应细胞接触时退出增殖细胞周期。我们在这里的重点是接触抑制作为一个例子的方法来诱导平静。以前的研究报告说, 细胞接触可以影响 rna 生物25, 因此, 结果提供了一个静止方法的信息, 需要额外的研究来确定哪些变化是与静止相关的本身。
我们使用的主要人二倍体成纤维细胞, 我们从新生儿皮肤分离。我们发起这些实验的纤维细胞传代少于10次。我们丢弃后通道成纤维细胞, 因为它们衰老。虽然本议定书的重点是增殖和静止真皮成纤维细胞, 所描述的程序可以用来监测衰变率在不同类型的细胞和在不同的条件。
我们使用转录关闭时间过程来监测基因组范围内的 mRNA 衰变率。关闭时间课程是最广泛使用的方法, 解耦 mrna 衰变的 mrna 转录, 以计算 mrna 半衰期1,26。在这里描述的方法, ActD, 一种药物, 与 B 型 DNA, 以阻止伸长的 RNA 聚合酶 II, 被用来逮捕转录的基因27。在8小时的时间内, 成绩单的数量减少的比率可以确定为成绩单的衰变率。
ActD-based 方法的一个重要的限制, 以监测成绩单衰变是, ActD 防止全球转录, 这将影响细胞的生存能力。在解释 ActD-based 数据时应牢记这一点, 因为衰变率是在比它们的原生状态更有压力的细胞中确定的。另一个重要的问题是, ActD 的影响可能不同的细胞在不同的状态, 包括增殖和接触抑制成纤维细胞。减少 ActD 治疗副作用的一种方法是保持时间课程短。因此, 我们选择了一个8小时的时间课程。
有其他的方法来 ActD 监测成绩单衰变率28,29。酵母菌株与温度敏感的 RNA 聚合酶 II 等位基因被用来监测成绩单衰变率30。这种方法的变化称为 "锚-客场" 已被用于酵母, 以达到条件耗尽 RNA 聚合酶 II 响应雷帕霉素治疗31,32。它也可以监测细胞的衰变与主动转录。例如, 硫代取代的嘧啶核苷酸可以被引入到细胞中, 加入到 mRNA 中, 随后检测到33,34,35。采用这种方法, 可以在继续合成 mRNA 的细胞中确定不同转录的合成速率。这种方法比 ActD 治疗有很大的优势, 因为它们不是有毒的。然而, 根据我们的经验, 这些方法在记录誊本的恢复过程中可能存在变化。ActD 治疗是一种直接和可重复的方法, 用于监测成绩单衰变。
所提供的协议中的几个步骤对其成功至关重要。一个重要的问题是在 rna 分离过程中防止 rna 降解。在步骤4.1 中, 应监测 RNA 的降解。如果 RNA 不包含18S 和 28S rRNAs 的两个清晰峰值 (图 4), 则表明已发生了成绩单降级。有些仪器提供 RNA 完整性数字 (林) 分数从1到10不等。在7-10 范围内的样品与林分评分是可以接受的进一步分析。如果 rna 降解, 那么重要的是采取更多的预防措施, 以防止 rna 的退化, 如确保解决方案和 sterileware 是无核糖核酸, 而且手套是佩戴时, 处理管的 rna。水可以用0.1% 二乙基焦 (DEPC) 进行预处理, 在37° c 下孵育至少2小时, 并且至少15分钟的蒸压. 请注意, DEPC 不能与 Tris-based 缓冲区一起使用。核糖核酸净化解决方案还可用于从工作表面、离心机和管中去除核糖核酸, 并可将抑制 RNases 的试剂添加到样品中。
另一个关键的步骤是分离的阶段, 苯酚-胍异硫氰酸酯溶液。重要的是要确保只有顶部相收集的 RNA 处理。如果产生的 RNA 包括剩余的苯酚, 230、260和 280 nm 的吸光度比值将不在 1:2: 1。高于预期的吸光度在 230 nm 可能表明苯酚污染。如果发生这种情况, RNA 可以再次沉淀, 再洗几次以除去残留的苯酚。
最后, 当 RNA 的颗粒和上清被删除, 颗粒可以粘稠, 薄, 容易受到干扰。在这一点上, 重要的是采取额外的照顾, 以脱下液体。它可能是重要的可视化管与良好的照明, 以找到球团, 并确保它不被打扰。添加糖原可以帮助确保颗粒在这一点上是可见的。
时间点的选择在衰变过程中也是一个重要的问题。虽然我们选择的四 timepoints 允许我们监测超过1万基因的衰变, 但有些基因在 8 h 时间过程中并没有明显的衰变。如果短命的成绩单是一个高优先级, 调查人员可能希望添加额外的样本收集之前, 120 分钟, 例如在30分钟。为了更好地评估长寿记录, 调查人员可能希望在以后的 timepoints 中添加更多的样本集。
如果衰变率不遵循预期的分布, 可能会产生另一个潜在问题。例如, 可能有太多或过少的基因显示对数线性衰变率超过8小时。如果发生这种情况, 它有助于监测已知的短或长寿基因的衰变率, 以确定它们是否遵循预期的模式。如果基因有一个积极的而不是消极的斜率随着时间的推移, 它们可以被排除在进一步的分析, 因为它们没有显示对数线性衰变率。如果被比较的两个条件中的细胞对 ActD 的反应有很大的不同, 那么许多基因在一个条件下就会衰变得更快。在这种情况下, 不仅要提到两国之间衰变率的差异, 而且还要参考我们建议的 spike-in 控制, 包括在 RNA 隔离之前, 以提供两种状态下的绝对衰变率的信息。
监测转录衰减率的能力, 在增殖和静止细胞或任何两个类似的国家有潜力进一步了解的重要性, 转录衰变在调节生理变化。应用包括具有和不致癌活化的细胞, 衰老或病毒感染之前和之后, 有和没有特定基因的击倒, 或在两种不同的分化状态。该研究结果可与 RNA 序列结合, 提供有关型的转录衰变率的特定变化的信息, 这可能提供洞察的变化, 型表达观察到细胞经历生理转变。

成绩单的稳定状态反映了成绩单合成和成绩单衰减的贡献。调节和协调转录衰减是控制生物过程的重要机制1,2,3,4。例如, 成绩单衰变率已被证明是贡献的时间序列事件后, 激活的炎症细胞因子肿瘤坏死因子5。
我们以前已经表明, 在原始人成纤维细胞的增殖和平静之间的过渡与许多基因的转录水平的变化有关6。其中一些变化反映了这两个国家之间转录因子活动的差异。
成绩单的衰变率的变化也会导致在两个不同状态7,8中的成绩单的表达水平的变化。根据我们早先的发现, 扩散与静止之间的过渡与多个 rna9的级别的变化有关, 我们询问是否也有来自差异成绩单衰减的贡献增殖与静止成纤维细胞的基因表达。
为了监测成绩单的稳定性, 我们确定了在增殖与静止细胞中转录的全基因组的衰变率。为了达到这个目的, 我们通过引入一种新的转录抑制剂和监测个体转录在一段时间内消失的速率来监测增殖与静止成纤维细胞的衰变率。这种方法的优点是, 与简单监控总体基因表达的方法相比, 通过抑制新的转录合成, 我们将能够分别从它们的速率来确定这些转录的衰变率。转录.
ActD 治疗, 以抑制新的转录和确定成绩单衰变率已成功地应用于以往的多项研究。ActD 已经被用来解剖的重要性, RNA 的稳定性在转录丰度变化的结果, 从治疗与炎细胞因子5。同样的方法也被用来解剖的差异, 转录衰变率在增殖与分化的 C2C12 细胞, 因为他们采用了分化肌肉表型10。另外一个例子, 在多能和分化的小鼠胚胎干细胞中也检测到了全球 mRNA 半衰期11。在这些例子中, mRNA 衰变已被证明是非常重要的调节转录丰度和细胞向不同的细胞状态转变。
应用下面所述的方法, 我们发现, 当比较成纤维细胞在增殖和静态状态12时, 大约500基因的转录衰变率发生了变化。特别是, 我们发现 rna miR-29(在静止单元中 downregulated) 的目标在单元格过渡到静止时稳定。我们在这里描述的方法, 我们用来确定衰变率在增殖和静止细胞。这一方法是有用的比较全球 mRNA 衰变率在两个截然不同的, 但类似的条件, 当信息有关迅速腐烂的基因寻求。它也可以用来解决其他问题, 如细胞培养条件对转录衰变的影响, 例如, 在二维与三维的文化。衰变率可以被确定的基因组范围内的方法, 如微阵列或 RNA 序列. 或者, real-time qPCR 或北印迹可以用来确定一个基因的衰变率或型的型基础。这些速率可以用来计算每个被监测基因的半衰期, 并在两种情况下确定具有不同衰变率的基因。

<table><tbody><tr><td>Centrifuges for microcentrifuge tubes capable of reaching 12,000 x g and 4&amp;deg;C</td><td></td><td></td><td></td></tr><tr><td>Equipment for running agarose gels</td><td></td><td></td><td></td></tr><tr><td>Sterile tissue culture plates and conical tubes</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Life Technologies</td><td>11965-118</td><td></td></tr><tr><td>Fetal bovine serum</td><td>VWR</td><td>35-010-CV</td><td></td></tr><tr><td>Sterile serological pipets, pipettors and pipet tips for tissue culture</td><td></td><td></td><td></td></tr><tr><td>Individually wrapped, disposable Rnase-free pipettes, pipette tips and tubes for RNA isolation and analysis</td><td></td><td></td><td></td></tr><tr><td>Disposable gloves to be worn when handling reagents and RNA samples</td><td></td><td></td><td></td></tr><tr><td>RNaseZap</td><td>Invitrogen</td><td>AM9780</td><td>For decontaminating work surfaces from RNase</td></tr><tr><td>2.0 ml eppendorf tubes</td><td></td><td></td><td></td></tr><tr><td>Trypsin-EDTA&amp;nbsp; Solution 10X</td><td>Millipore Sigma</td><td>9002-07-07</td><td></td></tr><tr><td>Sterile PBS</td><td>Life Technologies</td><td>14190-250</td><td></td></tr><tr><td>Trizol</td><td>Thermo Fisher Scientific</td><td>15596018</td><td></td></tr><tr><td>Actinomycin D</td><td>Millipore Sigma</td><td>A1410-2 mg</td><td>inhibits transcription</td></tr><tr><td>Sterile DMSO</td><td>Fisher Scientific</td><td>31-761-00ML</td><td>solvent for actinomycin D</td></tr><tr><td>Chloroform</td><td>Thermo Fisher Scientific</td><td>ICN19400225</td><td>MP Biomedicals, Inc product</td></tr><tr><td>Isopropanol</td><td>Fisher Scientific</td><td>BP2618500</td><td>molecular biology grade</td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>BP28184</td><td>molecular biology grade</td></tr><tr><td>RNase-free Glycogen (20 mg/ml aqueous solution)</td><td>Thermo Fisher Scientific</td><td>R0551</td><td>carrier for RNA precipitation</td></tr><tr><td>TURBO DNA-free Kit</td><td>Thermo Fisher Scientific</td><td>AM1907</td><td>removes DNA with DNase, then, in a subsequent step,&amp;nbsp; inactivates DNA and removes divalent cations</td></tr><tr><td>Agarose</td><td>Thermo Fisher Scientific</td><td>ICN820721</td><td>MP Biomedicals, Inc product</td></tr><tr><td>Loading dye for RNA gel</td><td>Thermo Fisher Scientific</td><td>R0641</td><td>suitable even for denaturing electrophoresis</td></tr><tr><td>Millenium RNA markers</td><td>Thermo Fisher Scientific</td><td>AM7150</td><td>RNA ladder</td></tr><tr><td>One-color RNA Spike-in Kit</td><td>Agilent Technology</td><td>5188-5282</td><td>Example spike-in control for Agient microarray analysis</td></tr><tr><td>Tris base</td><td>Fisher Scientific</td><td>BP152-1</td><td>molecular biology grade, for TAE buffer</td></tr><tr><td>Glacial acetic acid</td><td>Fisher Scientific</td><td>A38-500</td><td>for TAE buffer</td></tr><tr><td>EDTA</td><td>Fisher Scientific</td><td>BP120-500</td><td>electrophoresis grade, for gels</td></tr><tr><td>Ethidium bromide</td><td>Millipore Sigma</td><td>E7637</td><td>for molecular biology</td></tr><tr><td>External RNA Controls Consortium RNA Spike-in Mix</td><td>Thermo Fisher Scientific</td><td>4456740</td><td>Spike-in control for RNA Seq</td></tr><tr><td>TruSeq Stranded mRNA Library Preparation Kit A (48 samples, 12 indexes)</td><td>Illumina</td><td>RS-122-2101</td><td>for RNA Seq</td></tr><tr><td>96-well 0.3 ml PCR plate</td><td>Thermo Fisher Scientific</td><td>AB-0600</td><td>for real-time qPCR</td></tr><tr><td>Microseal B adhesive seals</td><td>Bio-Rad</td><td>MSB1001</td><td>for real-time qPCR</td></tr><tr><td>Rnase/Dnase-free Reagent Reservoirs</td><td>VWR</td><td>89094-662</td><td>for real-time qPCR</td></tr><tr><td>Rnase/Dnase-free Eight tube strips and caps</td><td>Thermo Fisher Scientific</td><td>AM12230</td><td>for real-time qPCR</td></tr><tr><td>SuperScript Reverse Transcriptase</td><td>Invitrogen</td><td>18090010</td><td>for real-time qPCR</td></tr><tr><td>AMPure XP Beads</td><td>Beckman Coulter</td><td>A63880</td><td>for real-time qPCR</td></tr></tbody></table>

determining genome-wide transcript decay rates in proliferating and quiescent human fibroblasts

静止是一种暂时的可逆状态, 其中细胞停止了细胞分裂, 但保留了增殖的能力。包括我们在内的多项研究表明, 静止与基因表达的广泛变化有关。其中一些变化发生在扩散相关转录因子的水平或活动的变化, 如 E2F 和 c-myc。我们已经证明, mRNA 的衰变也可以促进增殖和静止细胞之间基因表达的改变。在本协议中, 我们描述了建立人真皮包皮成纤维细胞增殖和静止培养的过程。然后, 我们描述的程序, 抑制新的转录在增殖和静止细胞与放线菌 D (ActD)。ActD 治疗是一种直接和可重复的方法, 游离新转录从成绩单衰变。ActD 治疗的一个缺点是, 由于 ActD 影响细胞的生存能力, 时间过程必须限制在短时间内。成绩单的水平被监测的时间, 以确定成绩单衰变率。这一过程允许鉴定的基因和异构体, 表现出差异衰变的增殖与静止成纤维细胞。

我们以前曾报道过在8小时的时间课程12中, 增殖和接触抑制主要人成纤维细胞的转录衰变率的微阵列分析结果。在补充表 1中提供了与增殖和接触抑制成纤维细胞比较的转录稳定性有显著变化的基因列表。荧光强度在时间零和在时间路线以后 ActD 治疗提供。基因被列入了名单上, 通过测定在增殖和接触抑制条件下有明显不同的斜坡的基因, 使用方差分析 f-试验。在图 5中显示了在增殖和静止成纤维细胞中有不同衰变率的基因的例子。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56423/56423fig1.jpg"> 
图 1: 用于建立 ActD 时间过程分析的增殖和接触抑制板的协议示意图.为 ActD 治疗提供细胞培养的必要步骤的一天描述, 假设四 timepoints 将被用于时间路线。为简单起见, 一个板块显示每 timepoint, 但额外的板块可以添加, 以产生生物复制。如果需要超过四 timepoints, 还可以调整该协议以添加更多的单元格。<a href="//ecsource.jove.com/files/ftp_upload/56423/56423fig1large.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/56423/56423fig2.jpg"> 
图 2: ActD 时间历程和衰变率计算的示意图与增殖的人成纤维细胞相比, 静态的.在使用转录抑制剂治疗后, 基因的预期分数会随着时间的推移而保留下来。下面, 在一段时间内残留的 mRNA 片段的样本被提供给在静态中比增殖细胞更稳定的基因, 以及在增殖中比静止细胞更稳定的基因。对于每个数据, 只显示单个值以进行清晰。在实际的协议中, 每个 timepoint 将有三数据。这一数字已由伊丽莎白 Pender 约翰逊的博士论文得到了她的同意。<a href="//ecsource.jove.com/files/ftp_upload/56423/56423fig2large.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/56423/56423fig3.jpg"> 
图 3: 在离心前后混合苯酚-胍异硫氰酸酯试剂和氯仿的照片.在混合后和离心后, 先将苯酚-胍异硫氰酸酯试剂和氯仿相结合, 进行混合。混合物离心后, 底部相是红色, 是有机相。中间的相间是白色和多云。顶部相是水相, 它是明确的。RNA 在水相, 应该收集用吸管为降雨雪。<a href="//ecsource.jove.com/files/ftp_upload/56423/56423fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56423/56423fig4.jpg"> 
图 4: 样品 rna 凝胶来监测 rna 的质量.用完整的 rna 显示样品的 rna 凝胶的例证。完整的 RNA 有突出的波段为28S 和 18S rRNAs。<a href="//ecsource.jove.com/files/ftp_upload/56423/56423fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56423/56423fig5.jpg"> 
图 5: 在增殖与静止成纤维细胞中有不同衰变率的基因的例子.荧光强度 (基因表达) 随着时间的推移显示的四基因衰变不同的增殖与静态成纤维细胞。<a href="//ecsource.jove.com/files/ftp_upload/56423/56423fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
补充表 1: 增殖和静止成纤维细胞的基因特异荧光强度。在 ActD 治疗增殖和接触抑制的成纤维细胞后, 提供了时间零和一段时间过程的数据。提供了 P 值、q 值和假发现率。表从BMC 基因组12中的原始出版物中重印。<a href="https://www.jove.com/files/ftp_upload/56423/Supplementary_Table_S1_080317.xlsx" target="_blank">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts at JoVE.com

Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts

我们描述了一种生成增殖和静止的主要人真皮成纤维细胞的协议, 监测转录衰变率, 并识别差异腐烂的基因。

增殖和静止人成纤维细胞的全基因组转录衰变率的测定

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ...

determining-genome-wide-transcript-decay-rates-proliferating

Nanyang Technological University

Research

JoVE Journal

Genetics

6.1K 次观看.  University of California, Los Angeles. 我们描述了一种生成增殖和静止的主要人真皮成纤维细胞的协议, 监测转录衰变率, 并识别差异腐烂的基因。

视频: Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

科研

遗传学

Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in production or degradation of RNA. This protocol describes a method for measurement of RNA production, using 5-Bromouridine labelling of RNA followed by immunoprecipitation, which enables investigation of RNA synthesized within a short timeframe (e.g., 1 h). The advantage of 5-Bromouridine-labelling and immunoprecipitation over the use of toxic transcriptional inhibitors, such as &#945;-amanitin and actinomycin D, is that there are no or very low effects on cell viability during short-term use. However, because 5-Bromouridine-immunoprecipitation only captures RNA produced within the short labelling time, slowly produced as well as rapidly degraded RNA can be difficult to measure by this method. The 5-Bromouridine-labelled RNA captured by 5-Bromouridine-immunoprecipitation can be analyzed by reverse transcription, quantitative polymerase chain reaction, and next generation sequencing. All types of RNA can be investigated, and the method is not limited to measuring mRNA as is presented in this example.

This method can be used to measure RNA synthesis. 5-Bromouridine is added to cells and incorporated into synthesized RNA. RNA synthesis is measured by RNA extraction immediately after labelling, followed by 5-Bromouridine-targeted immunoprecipitation of labelled RNA and analysis by reverse transcription and quantitative polymerase chain reaction.

用 5-Bromouridine 标记和免疫沉淀 RNA 合成的研究

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in ...

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 ...

Efficient Transcriptionally Controlled Plasmid Expression System for Investigation of the Stability of mRNA Transcripts in Primary Alveolar Epithelial Cells

Studying posttranscriptional regulation is fundamental to understanding the modulation of a given messenger RNA (mRNA) and its impact on cell homeostasis and metabolism. Indeed, fluctuations in transcript expression could modify the translation efficiency and ultimately the cellular activity of a transcript. Several experimental approaches have been developed to investigate the half-life of mRNA although some of these methods have limitations that prevent the proper study of posttranscriptional modulation. A promoter induction system can express a gene of interest under the control of a synthetic tetracycline-regulated promoter. This method allows the half-life estimation of a given mRNA under any experimental condition without disturbing cell homeostasis. One major drawback of this method is the necessity to transfect cells, which limits the use of this technique in isolated primary cells that are highly resistant to conventional transfection techniques. Alveolar epithelial cells in primary culture have been used extensively to study the cellular and molecular biology of the alveolar epithelium. The unique characteristics and phenotype of primary alveolar cells make it essential to study the posttranscriptional modulations of genes of interest in these cells. Therefore, our aim was to develop a novel tool to investigate the posttranscriptional modulations of mRNAs of interest in alveolar epithelial cells in primary culture. We designed a fast and efficient transient transfection protocol to insert a transcriptionally controlled plasmid expression system into primary alveolar epithelial cells. This cloning strategy, using a viral epitope to tag the construct, allows for the easy discrimination of construct expression from that of endogenous mRNAs. Using a modified &#916;&#916; quantification cycle (Cq) method, the expression of the transcript can then be quantified at different time intervals to measure its half-life. Our data demonstrate the efficiency of this novel approach in studying posttranscriptional regulation in various pathophysiological conditions in primary alveolar epithelial cells.

Here, we present a tool that can be used to study the posttranscriptional modulation of a transcript in primary alveolar epithelial cells by using an inducible expression system coupled to a pipette electroporation technique.

有效转录控制质粒表达系统，用于研究原发性奥子拉皮细胞中mRNA笔录稳定性

Studying posttranscriptional regulation is fundamental to understanding the modulation of a given messenger RNA (mRNA) and its impact on cell homeostasis ...

Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture

Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used approaches to determine protein half-life are either limited to population averages from lysed cells or require the use of protein synthesis inhibitors. This protocol describes a method to measure protein half-lives in single living adherent cells, using SNAP-tag fusion proteins in combination with fluorescence time-lapse microscopy. Any protein of interest fused to a SNAP-tag can be covalently bound by a fluorescent, cell permeable dye that is coupled to a benzylguanine derivative, and the decay of the labeled protein population can be monitored after washout of the residual dye. Subsequent cell tracking and quantification of the integrated fluorescence intensity over time results in an exponential decay curve for each tracked cell, allowing for determining protein degradation rates in single cells by curve fitting. This method provides an estimate for the heterogeneity of half-lives in a population of cultured cells, which cannot easily be assessed by other methods. The approach presented here is applicable to any type of cultured adherent cells expressing a protein of interest fused to a SNAP-tag. Here we use mouse embryonic stem (ES) cells grown on E-cadherin-coated cell culture plates to illustrate how single cell degradation rates of proteins with a broad range of half-lives can be determined.

This protocol describes a method to determine protein half-lives in single living adherent cells, using pulse labeling and fluorescence time-lapse imaging of SNAP-tag fusion proteins.

粘附细胞培养的时间推移荧光显微镜对蛋白质降解率的单细胞定量研究

Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used ...

发育生物学

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of the RNA produced during a brief period of labelling. This is done by attaching a biotin moiety to the incorporated thio group and affinity purifying, using streptavidin coated beads. Achieving a good yield of pure, newly synthesized RNA that is free of pre-existing RNA makes shorter labelling times possible and permits increased temporal resolution in kinetic studies. This is a protocol for very specific, high yield purification of newly synthesized RNA. The protocol presented here describes how RNA is extracted from the yeast Saccharomyces cerevisiae. However, the protocol for purification of thiolated RNA from total RNA should be effective using RNA from any organism once it has been extracted from the cells. The purified RNA is suitable for analysis by many widely used techniques, such as reverse transcriptase-qPCR, RNA-seq and SLAM-seq. The specificity, sensitivity and flexibility of this technique allow unparalleled insights into RNA metabolism.

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil Ers4tU

The use of thiolated uracil to sensitively and specifically purify newly transcribed RNA from the yeast Saccharomyces cerevisiae.

极快速和特殊的代谢标签的RNA在Vivo与4-Thiouracil （Ers4tU）

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of ...

生物化学

Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as Alzheimer&#39;s and Parkinson&#39;s diseases. Cellular senescence is a state of permanent cell cycle arrest that typically occurs in response to exposure to sub-lethal stresses. However, like other non-dividing cells, senescent cells remain metabolically active and carry out many functions that require unique transcriptional and translational demands and widespread changes in the intracellular and secreted proteomes. Understanding how protein synthesis and decay rates change during senescence can illuminate the underlying mechanisms of cellular senescence and find potential therapeutic avenues for diseases exacerbated by senescent cells. This paper describes a method for proteome-scale evaluation of protein half-lives in non-dividing cells using pulsed stable isotope labeling by amino acids in cell culture (pSILAC) in combination with mass spectrometry. pSILAC involves metabolic labeling of cells with stable heavy isotope-containing versions of amino acids. Coupled with modern mass spectrometry approaches, pSILAC enables the measurement of protein turnover of hundreds or thousands of proteins in complex mixtures. After metabolic labeling, the turnover dynamics of proteins can be determined based on the relative enrichment of heavy isotopes in peptides detected by mass spectrometry. In this protocol, a workflow is described for the generation of senescent fibroblast cultures and similarly arrested quiescent fibroblasts, as well as a simplified, single-time point pSILAC labeling time-course that maximizes coverage of anticipated protein turnover rates. Further, a pipeline is presented for the analysis of pSILAC mass spectrometry data and user-friendly calculation of protein degradation rates using spreadsheets. The application of this protocol can be extended beyond senescent cells to any non-dividing cultured cells such as neurons.

This protocol describes the workflow for metabolic labeling of senescent and non-dividing cells with pulsed SILAC, untargeted mass spectrometry analysis, and a streamlined calculation of protein half-lives.

使用代谢标记和质谱法测量衰老和非分裂培养细胞中的蛋白质周转率

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as ...

Measuring the Kinetics of mRNA Transcription in Single Living Cells

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo is of importance. Pol II kinetics have been measured using biochemical or molecular methods.1-3 In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells.4 Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells.5, 6 Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo, it is possible to detect the actual transcription of mRNAs on the gene of interest.7, 8 The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed.9 To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs.5 In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.

RNA polymerase II transcriptional kinetics are measured on specific genes in living cells. mRNAs transcribed from the gene of interest are fluorescently tagged and using Fluorescence Recovery After Photobleaching (FRAP) the in vivo kinetics of transcriptional elongation are obtained.

测量单个活细胞中的mRNA转录的动力学

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

生物学

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e. total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated.
We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.

Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.

高分辨率基因表达谱RNA的合成，加工和衰变的新转录RNA在细胞培养的代谢标记

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene ...

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Mutation rates in young Saccharomyces cerevisiae cells measured through fluctuation tests are used to predict mutation frequencies for mother cells of different replicative ages. Magnetic sorting and flow cytometry are then used to measure actual mutation frequencies and age of mother cells to identify any deviations from predicted mutation frequencies.

结合母细胞和波动测试磁分选，以分析基因组不稳定性的过程中有丝分裂细胞衰老<em&gt;酿酒酵母</em

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this ...