Isolation of Small Noncoding RNAs from Human Serum
Isolation of Small Noncoding RNAs from Human Serum
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DOI：
10.3791/51443
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发表时间：
2014-06-01
影响因子：
1.2
通讯作者：
Nham Tran
中科院分区：
文献类型：
--
作者：
Khoury, Samantha;Ajuyah, Pamela;Nham Tran
RNA及其表达的分析是许多实验室的共同特征。重要的是小RNA的出现，如在哺乳动物细胞中发现的microRNA。这些小RNA是控制重要途径如生长、发育和死亡的有效基因调节剂，并且已经针对它们在体液中的表达产生了很多兴趣。这是由于它们在人类疾病如癌症中的失调以及它们作为血清生物标志物的潜在应用。然而，血清中miRNA表达的分析可能存在问题。在大多数情况下，血清的量是有限的，血清含有少量的总RNA，其中小RNA仅占0.4-0.5%（1）。因此，从血清中分离足够量的优质RNA是当今研究人员面临的主要挑战。在这篇技术论文中，我们展示了一种方法，该方法仅使用400 μ l的人血清来获得足够的RNA用于DNA阵列或qPCR分析。该方法的优点是其简单性和产生高质量RNA的能力。它不需要专门的柱来纯化小RNA，并利用常见实验室中的通用试剂和硬件。我们的方法利用锁相凝胶来消除苯酚污染，同时产生高质量的RNA。我们还引入了一个额外的步骤，以进一步去除分离步骤中的所有污染物。该方案在从血清中分离高达100 ng/μ l的总RNA产量方面非常有效，但也可适用于其他生物组织。
The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%(1). Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 mu l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/mu l from serum but can also be adapted for other biological tissues.