Name: profiling individual human embryonic stem cells by quantitative rt-pcr
Uploaded: 2014-05-29T13:00:00
Description: Watch this Scientific Journal Video about Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR at JoVE.com

我们要感谢李实验室的成员对稿件有价值的讨论。在李的实验室工作是由纽约干细胞基金会罗伯逊研究奖和美国马里兰干细胞研究基金（TEDCO）资助。

一个96孔板的1。下游<ol><li>混合1μL单细胞DNase1至9微升单细胞裂解液。 </li><li>把10μl的混合溶液中的96孔PCR板各孔中。 </li></ol> 2，拆卸人类胚胎干细胞的分离纯化流式细胞仪<ol><li>从60毫米培养皿用1ml的Accutase 20分钟，在37℃，这是中和人ES介质分离OCT4 :: EGFP的胚胎干细胞系。 </li><li>制备的细胞群体在1ml的FACS缓冲液和细胞调整至1×10 6细胞/ ml。 </li><li>通过一个35微?...

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单细胞基因分析可能是一个主要的工具来预测一个单细胞的功能或整个人口。由于技术的限制，全基因谱分析已经被限制人口的平均水平。中的基因表达模式和个体的细胞和亚群之间的水平的变化已经被提出来引起错误的解释。这些不同的细胞方面可以在人类胚胎干细胞被发现和他们的异质性造成微妙的不同能力维持多能性和命运规范的过程。 我们开发并验证了单细胞定量RT...

大多数高等真核生物的人群是异质从而汇集与人口分析，它往往是难以解释其细胞的功能。在一个人口单个细胞可能是细微的差别，而这些差别能有重要影响整个人群1，2的特性和功能。特别是，人胚胎干细胞（胚胎干细胞）被称为是异质的，这会导致不同程度的多能性和多样化的电位，以微妙独特的方式3，4谱系规范。例如，不同的细胞表面抗原，可用于分类的未分化的多能干细胞，5和奥斯汀史密斯组建议在小鼠胚胎干细胞不同级别的多能性，根据其形态，分化倾向和信号转导通路6的依赖性。这种现象被推测在人类胚胎NIC干细胞7。而整体的研究，不同的干细胞系，而不是单个单个干细胞中进行的，它可能是非常有趣的，分析不同层次的多能性的在单细胞水平，这可能会影响其分化能力，向全体细胞谱系。 细胞和分子异质性可以通过转录分析来决定的，这就是所谓的“单细胞转录组”，并强调定量基因表达水平8-10新方法。用于在单个细胞的基因表达水平的分析，我们开发了单细胞定量RT-PCR的一个简单的，但健壮的协议。我们证实了我们的协议的有效性和可行性通过比较单个细胞裂解物各一半以及人类胚胎干细胞的连续稀释的总RNA，产生最小的技术的变化和差异。进一步，我们使用了遗传记者线隔离同质population利用基因打靶系统的人类胚胎干细胞。用于靶向OCT4基因座（OCT4-2A-EGFP-PGK-迪普 ​​罗构造）和一对TALEN质粒的供体载体被用来11。供体载体和一对TALEN质粒导入人类胚胎干细胞（H9，WA09）使用我们的核转染和克隆选择协议和维护人类胚胎干细胞是基于我们的日常协议12进行的。我们证实了这OCT4基因记者一行表达绿色荧光蛋白的表达OCT4 :: EGFP的胚胎干细胞。 我们的结果表明，个体的胚胎干细胞（排序OCT4 :: EGFP强阳性细胞）保持高水平的OCT4的表达，但不同层次的NANOG的表达。所以，我们的单细胞基因表达分析应该对学习多能干细胞的人口异质性是有用的。

<table border="0" cellpadding="0" cellspacing="0" width="807">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">96 wll PCR Plate</td>
			<td style="width:123px;">USA scientific</td>
			<td style="width:119px;">1402-8900</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Ambion Cell Lysis Kit</td>
			<td style="width:123px;">Life Technologies</td>
			<td align="right" style="width:119px;">4458235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">SMARTScribe Reverse Transcriptase</td>
			<td style="width:123px;">Clonetech</td>
			<td align="right" style="width:119px;">639536</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">ExoSAP-IT</td>
			<td style="width:123px;">USB</td>
			<td align="right" style="width:119px;">78200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Platinum Taq DNA polymerase High Fidelity</td>
			<td style="width:123px;">Invitrogen</td>
			<td align="right" style="width:119px;">11304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">10mM dNTP Mix, PCR Grade</td>
			<td style="width:123px;">Invitrogen</td>
			<td align="right" style="width:119px;">18427</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">SYBR Universal 2X Master Mix</td>
			<td style="width:123px;">Kapa biosystem</td>
			<td style="width:119px;">KR0389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Accutase</td>
			<td>Innovative Cell Tech</td>
			<td>S-1100-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">FACS buffer</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td>45 ml PBS, 5 ml a-MEM, 100 ul DNase, filter sterilized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">35 &#956;m cell strainer cap tubes</td>
			<td style="width:123px;">BD Biosciences</td>
			<td align="right" style="width:119px;">352235</td>
			<td></td>
		</tr>
	</tbody>
</table>

profiling individual human embryonic stem cells by quantitative rt-pcr

干细胞群的异质性阻碍了干细胞生物学的详细了解，如他们的分化倾向朝着不同的谱系。单细胞转录组分析可以为解剖的个体差异的新方法。我们已经开发了单细胞定量RT-PCR法，并证实了该方法在几个基因表达谱效果很好。在单细胞水平上，每一个人类胚胎干细胞，按OCT4 :: EGFP阳性细胞，具有较高的表达OCT4，但不同级别NANOG的表达。我们的单细胞基因表达分析应该是有用的审问人口异质性。

高效率和强大的单细胞RNA扩增 为了尽量减少人类胚胎干细胞中的转录变化，我们使用的OCT4 :: EGFP的胚胎干细胞克隆流式细胞仪净化。 OCT4 :: EGFP阳性细胞分选入96孔板后，每个小区被裂解在裂解缓冲液中，并用SMA-T15（GACATGTATCCGGATGTTTTTTTTTTTTTTTT）引物和具有SMA-A（ACATGTATCCGGATGTGGG锚固转换的poly（A）+ RNA到cDNA全长）通过使用智能模板切换技术。多余的寡核苷酸?...

Watch this Scientific Journal Video about Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR at JoVE.com

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR (Video) | JoVE

分析单个人类胚胎干细胞通过定量RT-PCR

单细胞基因表达分析是需要了解干细胞的异质性。

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

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