In vitro application of ribonucleases: comparison of the effects on mRNA and miRNA stability.
In vitro application of ribonucleases: comparison of the effects on mRNA and miRNA stability.
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DOI：
10.1186/s13104-015-1114-z
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发表时间：
2015-04-22
影响因子：
1.8
通讯作者：
Denecke B
中科院分区：
文献类型：
--
作者：
Aryani A;Denecke B
MicroRNA在广泛的研究领域中变得非常重要。由于已知microRNA的数量不断增加，这些分子可能越来越多地被视为一类新的生物标志物。这是由microRNA在血浆中循环时相对稳定的事实驱动的。尽管对microRNA加工机制进行了广泛的分析，但对microRNA在特定条件下的体外衰变或mRNA和microRNA的相对稳定性知之甚少。在该体外研究中，在规定的条件下，用不同的核糖核酸酶处理相同RNA池的等量总RNA。使用主要基于核糖体RNA的微流体分析评估总RNA的降解。为了评价特异性RNA酶对不同种类的RNA（核糖体RNA、mRNA、miRNA）的影响，使用RT-qPCR测定法定量核糖体RNA以及特异性mRNA和miRNA的模式。通过与未处理的对照样品进行比较，确定了取决于RNA类别的核糖核酸酶特异性降解等级。在目前的体外研究中，我们研究了mRNA和microRNA的稳定性对实验室实践中使用的核糖核酸酶的影响。用特异性核糖核酸酶处理总RNA，并通过RT-qPCR和小型化凝胶电泳分析不同种类RNA的衰变。此外，我们还研究了核糖体RNA观察到的完整性是否适用于microRNA和mRNA。根据所使用的核糖核酸酶的种类，我们的结果表明，microRNA相对于mRNA具有更高的稳定性，并且核糖体RNA完整性与其他RNA组完整性的相关性有限。我们的研究结果表明，核糖体RNA的降解状态并不总是适用于mRNA和microRNA。事实上，这些RNA类暴露于核糖核酸酶的稳定性是相互独立的，microRNA比mRNA更稳定。microRNA的相对稳定性支持它们作为生物标志物在一系列应用中的潜力和进一步发展。
MicroRNA has become important in a wide range of research interests. Due to the increasing number of known microRNAs, these molecules are likely to be increasingly seen as a new class of biomarkers. This is driven by the fact that microRNAs are relatively stable when circulating in the plasma. Despite extensive analysis of mechanisms involved in microRNA processing, relatively little is known about the in vitro decay of microRNAs under defined conditions or about the relative stabilities of mRNAs and microRNAs. In this in vitro study, equal amounts of total RNA of identical RNA pools were treated with different ribonucleases under defined conditions. Degradation of total RNA was assessed using microfluidic analysis mainly based on ribosomal RNA. To evaluate the influence of the specific RNases on the different classes of RNA (ribosomal RNA, mRNA, miRNA) ribosomal RNA as well as a pattern of specific mRNAs and miRNAs was quantified using RT-qPCR assays. By comparison to the untreated control sample the ribonuclease-specific degradation grade depending on the RNA class was determined. In the present in vitro study we have investigated the stabilities of mRNA and microRNA with respect to the influence of ribonucleases used in laboratory practice. Total RNA was treated with specific ribonucleases and the decay of different kinds of RNA was analysed by RT-qPCR and miniaturized gel electrophoresis. In addition, we have examined whether the integrity observed for ribosomal RNA is applicable to microRNA and mRNA. Depending on the kind of ribonuclease used, our results demonstrated a higher stability of microRNA relative to mRNA and a limitation of the relevance of ribosomal RNA integrity to the integrity of other RNA groups. Our results suggest that the degradation status of ribosomal RNA is not always applicable to mRNA and microRNA. In fact, the stabilities of these RNA classes to exposure to ribonucleases are independent from each other, with microRNA being more stable than mRNA. The relative stability of microRNAs supports their potential and further development as biomarkers in a range of applications.