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...

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。

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	<tbody>
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			<td height="42" style="height:42px;width:291px;">Centrifuges for microcentrifuge tubes capable of reaching 12,000 x g and 4&#176;C</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td style="width:237px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Equipment for running agarose gels</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td style="width:237px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Sterile tissue culture plates and conical tubes</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td style="width:237px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:163px;">Life Technologies</td>
			<td style="width:119px;">11965-118</td>
			<td style="width:237px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Fetal bovine serum</td>
			<td style="width:163px;">VWR</td>
			<td style="width:119px;">35-010-CV</td>
			<td style="width:237px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Sterile serological pipets, pipettors and pipet tips for tissue culture</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td style="width:237px;"></td>
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			<td height="63" style="height:63px;width:291px;">Individually wrapped, disposable Rnase-free pipettes, pipette tips and tubes for RNA isolation and analysis</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td style="width:237px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Disposable gloves to be worn when handling reagents and RNA samples</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td style="width:237px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:291px;">RNaseZap</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:119px;">AM9780</td>
			<td style="width:237px;">For decontaminating work surfaces from RNase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">2.0 ml eppendorf tubes</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td style="width:237px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Trypsin-EDTA&#160; Solution 10X</td>
			<td style="width:163px;">Millipore Sigma</td>
			<td style="width:119px;">9002-07-07</td>
			<td style="width:237px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:291px;">Sterile PBS</td>
			<td style="width:163px;">Life Technologies</td>
			<td style="width:119px;">14190-250</td>
			<td style="width:237px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Trizol</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15596018</td>
			<td style="width:237px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Actinomycin D</td>
			<td style="width:163px;">Millipore Sigma</td>
			<td style="width:119px;">A1410-2 mg</td>
			<td style="width:237px;">inhibits transcription</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Sterile DMSO</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">31-761-00ML</td>
			<td style="width:237px;">solvent for actinomycin D</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Chloroform</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">ICN19400225</td>
			<td style="width:237px;">MP Biomedicals, Inc product</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Isopropanol</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">BP2618500</td>
			<td style="width:237px;">molecular biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Ethanol</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">BP28184</td>
			<td style="width:237px;">molecular biology grade</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">RNase-free Glycogen (20 mg/ml aqueous solution)</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">R0551</td>
			<td style="width:237px;">carrier for RNA precipitation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:291px;">TURBO DNA-free Kit</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AM1907</td>
			<td style="width:237px;">removes DNA with DNase, then, in a subsequent step,&#160; inactivates DNA and removes divalent cations</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Agarose</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">ICN820721</td>
			<td style="width:237px;">MP Biomedicals, Inc product</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Loading dye for RNA gel</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">R0641</td>
			<td style="width:237px;">suitable even for denaturing electrophoresis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Millenium RNA markers</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AM7150</td>
			<td style="width:237px;">RNA ladder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">One-color RNA Spike-in Kit</td>
			<td style="width:163px;">Agilent Technology</td>
			<td style="width:119px;">5188-5282</td>
			<td style="width:237px;">Example spike-in control for Agient microarray analysis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Tris base</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">BP152-1</td>
			<td style="width:237px;">molecular biology grade, for TAE buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Glacial acetic acid</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">A38-500</td>
			<td style="width:237px;">for TAE buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">EDTA</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">BP120-500</td>
			<td style="width:237px;">electrophoresis grade, for gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Ethidium bromide</td>
			<td style="width:163px;">Millipore Sigma</td>
			<td style="width:119px;">E7637</td>
			<td style="width:237px;">for molecular biology</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">External RNA Controls Consortium RNA Spike-in Mix</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td>4456740</td>
			<td style="width:237px;">Spike-in control for RNA Seq</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">TruSeq Stranded mRNA Library Preparation Kit A (48 samples, 12 indexes)</td>
			<td style="width:163px;">Illumina</td>
			<td style="width:119px;">RS-122-2101</td>
			<td style="width:237px;">for RNA Seq</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">96-well 0.3 ml PCR plate</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AB-0600</td>
			<td style="width:237px;">for real-time qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Microseal B adhesive seals</td>
			<td style="width:163px;">Bio-Rad</td>
			<td style="width:119px;">MSB1001</td>
			<td style="width:237px;">for real-time qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Rnase/Dnase-free Reagent Reservoirs</td>
			<td style="width:163px;">VWR</td>
			<td style="width:119px;">89094-662</td>
			<td style="width:237px;">for real-time qPCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Rnase/Dnase-free Eight tube strips and caps</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AM12230</td>
			<td style="width:237px;">for real-time qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">SuperScript Reverse Transcriptase</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:119px;">18090010</td>
			<td style="width:237px;">for real-time qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">AMPure XP Beads</td>
			<td style="width:163px;">Beckman Coulter</td>
			<td style="width:119px;">A63880</td>
			<td style="width:237px;">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...

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

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

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

在新鲜分离的人代绒毛滋养细胞转染和EMSA分析

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

科研

遗传学

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

在人原代细胞培养生物钟基因表达及荷尔蒙分泌的并行测量

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge.
This article describes in detail the &#34;Copy Number Ratio of S/G1 for mapping genomic Time of Replication&#34; method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

通过DNA含量测定的哺乳动物复制时序的全基因组的测定

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

与ChEC-seq的蛋白质-DNA相互作用的全基因组映射<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions of chromatin. These regions represent regulatory DNA elements (e.g., promoters, enhancers, locus control regions, insulators) to which transcription factors bind. Mapping the accessible chromatin landscape is a powerful approach for uncovering active regulatory elements across the genome. This information serves as an unbiased approach for discovering the network of relevant transcription factors and mechanisms of chromatin structure that govern gene expression programs. ATAC-seq is a robust and sensitive alternative to DNase I hypersensitivity analysis coupled with next-generation sequencing (DNase-seq) and formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) for genome-wide analysis of chromatin accessibility and to the sequencing of micrococcal nuclease-sensitive sites (MNase-seq) to determine nucleosome positioning. We present a detailed ATAC-seq protocol optimized for human primary immune cells i.e. CD4+ lymphocytes (T helper 1 (Th1) and Th2 cells). This comprehensive protocol begins with cell harvest, then describes the molecular procedure of chromatin tagmentation, sample preparation for next-generation sequencing, and also includes methods and considerations for the computational analyses used to interpret the results. Moreover, to save time and money, we introduced quality control measures to assess the ATAC-seq library prior to sequencing. Importantly, the principles presented in this protocol allow its adaptation to other human immune and non-immune primary cells and cell lines. These guidelines will also be useful for laboratories which are not proficient with next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin coupled with high-throughput sequencing (ATAC-seq) is a genome-wide method to uncover accessible chromatin. This is a step-by-step ATAC-seq protocol, from molecular to the final computational analysis, optimized for human lymphocytes (Th1/Th2). This protocol can be adopted by researchers without prior experience in next-generation sequencing methods.

ATAC 基因组在原发性人类 T 淋巴细胞中的染色体全易接近染色质

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions ...

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a protocol for performing the chromatin conformation capture technique in situ Hi-C on staged Drosophila melanogaster embryo populations. The result is a sequencing library that allows the mapping of all chromatin interactions that occur in the nucleus in a single experiment. Embryo sorting is done manually using a fluorescent stereo microscope and a transgenic fly line containing a nuclear marker. Using this technique, embryo populations from each nuclear division cycle, and with defined cell cycle status, can be obtained with very high purity. The protocol may also be adapted to sort older embryos beyond gastrulation. Sorted embryos are used as inputs for in situ Hi-C. All experiments, including sequencing library preparation, can be completed in five days. The protocol has low input requirements and works reliably using 20 blastoderm stage embryos as input material. The end result is a sequencing library for next generation sequencing. After sequencing, the data can be processed into genome-wide chromatin interaction maps that can be analyzed using a wide range of available tools to gain information about topologically associating domain (TAD) structure, chromatin loops, and chromatin compartments during Drosophila development.

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

This work describes a protocol for the generation of high resolution in situ Hi-C libraries from tightly staged pre-gastrulation Drosophila melanogaster embryos.

大范围早期果蝇胚胎的全基因组染色质构象捕获库的生成

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a ...

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the spatio-temporal expression of their target genes through physical contact, often bridging considerable (in some cases hundreds of kilobases) genomic distances and bypassing nearby genes. The human genome harbors an estimated one million enhancers, the vast majority of which have unknown gene targets. Assigning distal regulatory regions to their target genes is thus crucial to understand gene expression control. We developed Promoter Capture Hi-C (PCHi-C) to enable the genome-wide detection of distal promoter-interacting regions (PIRs), for all promoters in a single experiment. In PCHi-C, highly complex Hi-C libraries are specifically enriched for promoter sequences through in-solution hybrid selection with thousands of biotinylated RNA baits complementary to the ends of all promoter-containing restriction fragments. The aim is to then pull-down promoter sequences and their frequent interaction partners such as enhancers and other potential regulatory elements. After high-throughput paired-end sequencing, a statistical test is applied to each promoter-ligated restriction fragment to identify significant PIRs at the restriction fragment level. We have used PCHi-C to generate an atlas of long-range promoter interactions in dozens of human and mouse cell types. These promoter interactome maps have contributed to a greater understanding of mammalian gene expression control by assigning putative regulatory regions to their target genes and revealing preferential spatial promoter-promoter interaction networks. This information also has high relevance to understanding human genetic disease and the identification of potential disease genes, by linking non-coding disease-associated sequence variants in or near control sequences to their target genes.

DNA regulatory elements, such as enhancers, control gene expression by physically contacting target gene promoters, often through long-range chromosomal interactions spanning large genomic distances. Promoter Capture Hi-C (PCHi-C) identifies significant interactions between promoters and distal regions, enabling the assignment of potential regulatory sequences to their target genes.

启动子捕获高分辨率, 全基因组的启动子交互分析

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

全基因组对真核生物转录误差的监测

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

用细胞遗传学技术测定芽孢杆菌的受精率

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished.
Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

荧光活化细胞分选分离人皮肤状和网状成纤维细胞

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have ...