Name: isolation of small noncoding rnas from human serum
Uploaded: 2014-06-19T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Small Noncoding RNAs from Human Serum at JoVE.com

萨曼莎·扈利和Pamela Ajuyah由澳洲研究生奖支持。我们还要承认平移癌症研究网络，洛伊癌症研究中心，新南威尔士大学和北平移癌症研究单位对他们的额外支持萨曼莎·扈利的。

注：从健康的病人或癌症患者的人血清样品受到来自皇家阿尔弗雷德王子医院悉尼（协议号X10-0016和HREC/10/RPAH/24）和科技大学批准人的道德协议知情同意获得，悉尼。血清样品从患者在手术前，从各个悉尼医院收集和放置在存储于-80℃。 从血清1。小RNA分离总RNA，从人血清中使用三试剂RT LS协议的修改版本来制备。 <ol><li>解冻冷冻的血清样品在冰上，再换...

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microRNA的人口构成中的血清中发现的总RNA约0.4-0.5％。此外还有人血清中发现了蛋白质含量高。以提高RNA和减少蛋白质和酚污染我们修改了传统的Chomczynski方法9与另外的几个步骤。 总RNA从血清使用标准的三试剂RT-LS（分子研究中心）分离但是在我们的实验室中进行时，这种方法在低量含有各种杂质，得到的RNA。 图1显示了蛋白污染的证...

近年来，出现了越来越多的推动，发现新的生物标志物的早期检测人类疾病。许多注意力都集中在利用小分子RNA，如微RNA 2（微RNA或大鹏）作为潜在的标志物。这些小RNA被发现在体液如血清和研究已经表明它们是弹性的退化，并稳定在一个范围内的不同的环境条件3。鉴于这些特点，血清或循环miRNA是理想的生物标志物4，5。目前，有用于从生物体液的小RNA分离两种主要方法。第一种方法使用了基于列的技术结合和洗脱的小RNA 6，而第二种方法使用与苯酚和异硫氰酸胍试剂7已久的协议。我们已经开发出一种简单，有效，无柱协议从人血清中分离出的小分子RNA。分离的RNA是immediately可用在下游应用，包括DNA寡核苷酸阵列和RNA测序。 这个协议被开发，因为我们使用酚为基础的方法，从血清中分离RNA时，面临着几个问题。传统Chomczynski方法被经常使用在大多数实验室用范围可从大多数商业供应商的试剂。但是考虑到它们的广泛使用，严格的标准还未发展到持续生产高品质的RNA的体液，特别是血液或血清。 从血清中分离RNA相关的常见问题包括低的RNA产率和污染的隔离，特别是苯酚过程中使用的试剂。我们的方法消除了这些酚类污染物为下游分析，如定量PCR（定量PCR）和RNA测序提供高品质的RNA。我们对miRNA的阵列进一步测试此RNA。

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			<td height="22" style="height:22px;width:259px;">Name of the Material/Equipment&#160;</td>
			<td style="width:223px;">Company&#160;</td>
			<td style="width:123px;">Catalog Number&#160;</td>
			<td style="width:248px;">Comments/ Description (optional)&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Tri-Reagent RT LS</td>
			<td style="width:223px;">Molecular Research Centre, USA</td>
			<td style="width:123px;">TR 118</td>
			<td style="width:248px;">-</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:259px;">RNase free H20</td>
			<td style="width:223px;">GIBCO Invitrogen</td>
			<td style="width:123px;">10977-023</td>
			<td style="width:248px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Proteinase K&#160;</td>
			<td style="width:223px;">Finnzymes, Finland</td>
			<td style="width:123px;">EO0491</td>
			<td style="width:248px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Heavy Phaselock Tube</td>
			<td style="width:223px;">5PRIME</td>
			<td>2302830</td>
			<td style="width:248px;">2ml capacity</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">DNA Lobind tube</td>
			<td style="width:223px;">Eppendorf</td>
			<td style="width:123px;">0030 108.078</td>
			<td style="width:248px;">1.5ml capacity</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Glycogen&#160;</td>
			<td style="width:223px;">Invitrogen, USA</td>
			<td style="width:123px;">10814-010</td>
			<td style="width:248px;">5mg/ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">RNA grade Isopropanol&#160;</td>
			<td style="width:223px;">Sigma Aldrich, USA</td>
			<td style="width:123px;">I9516</td>
			<td style="width:248px;">100%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Refrigerated Centrifuge</td>
			<td style="width:223px;">John Morris</td>
			<td style="width:123px;">-</td>
			<td style="width:248px;">-</td>
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			<td height="22" style="height:22px;width:259px;">Nanodrop UV-Vis spectrophotometer</td>
			<td style="width:223px;">Thermo Fisher Scientific, USA</td>
			<td style="width:123px;">-</td>
			<td style="width:248px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">RNA grade Ethanol</td>
			<td style="width:223px;">Sigma Aldrich, USA</td>
			<td style="width:123px;">E7023</td>
			<td style="width:248px;">70%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Agilent 2100 Bioanalyser</td>
			<td style="width:223px;">Agilent, USA</td>
			<td style="width:123px;">-</td>
			<td style="width:248px;">-</td>
		</tr>
	</tbody>
</table>

isolation of small noncoding rnas from human serum

RNA和其表达的分析是在许多实验室的共同特征。意义的是小RNA像微小RNA，其被发现在哺乳动物细胞中的出现。这些小RNA是有效的调节基因控制的重要途径，如生长，发育和死亡，并极大的兴趣已经冲着他们的体液中的表达。这是由于它们在人类疾病如癌症和其潜在的应用如血清生物标志物的失调。然而，血清中的miRNA表达的分析可能会产生问题。在大多数情况下，血清的量限制和血清中含有低量的总RNA，其中小RNA只占0.4-0.5％1。因此足量的血清RNA的质量分离是当今的一大挑战研究人员。在这种技术论文，我们证明仅使用400μL人血清中获得足够的RNA是DNA阵列或定量PCR分析的方法。在一个这种方法的dvantages是它的简单性和产生高质量RNA的能力。它不需要专门的列的小RNA纯化及利用一般的试剂和硬件共同实验室发现。我们的方法是利用一个相位锁定凝胶消除苯酚污染，而在同一时间得到高质量的RNA。我们还引入一个额外的步骤在分离步骤，以进一步去除所有污染物。这个协议是非常有效的隔离血清高达100纳克/微升的总RNA的产率，但也可以适用于其他生物组织。

图1表示从血清中分离的RNA的典型的UV / Vis光谱。从这个配置文件，我们注意到蛋白质污染在无论是在270 nm和230 nm的280 nm的苯酚和有机污染物，分别。残留硫氰酸胍还注意到在260 nm处。以减少污染物，一系列的优化步骤是标准的三试剂RT-LS方法制备。我们添加了糖原的5毫克/毫升，以增加两总RNA的产量和减少污染（黑线， 图1A）。 进一步，我们观察到?...

Watch this Scientific Journal Video about Isolation of Small Noncoding RNAs from Human Serum at JoVE.com

Isolation of Small Noncoding RNAs from Human Serum

本协议描述为从人血清中提取的小分子RNA的方法。我们已经用这种方法来隔离患者血清小分子RNA在DNA阵列使用，也可单重定量PCR。该协议利用酚和异硫氰酸胍试剂进行了修改，以产生高质量的RNA。

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To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

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