Validation of miRNA genes suitable as reference genes in qPCR analyses of miRNA gene expression in Atlantic salmon (Salmo salar).
Validation of miRNA genes suitable as reference genes in qPCR analyses of miRNA gene expression in Atlantic salmon (Salmo salar).
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DOI：
10.1186/1756-0500-7-945
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发表时间：
2014-12-23
影响因子：
1.8
通讯作者：
Andreassen R
中科院分区：
文献类型：
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作者：
Johansen I;Andreassen R
microRNA（miRNAs）是一类丰富的内源性小RNA分子，其在转录后水平下调基因表达。它们通过调节控制多种生物学过程的基因发挥重要作用，近年来对miRNA基因和miRNA基因表达的研究兴趣增加。应用于研究单个基因的基因表达的最常见方法是定量PCR（qPCR）。然而，在研究成熟miRNA的表达之前，必须开发稳健的qPCR方法（miRNA-qPCR）。这包括鉴定和验证合适的参考基因。我们对大西洋鲑鱼（萨尔莫salar）特别感兴趣。这是一个经济上重要的水产养殖物种，但没有专门用于该物种的miRNA-qPCR方法的参考基因已经过验证。因此，我们的目标是在萨尔莫盐沼中鉴定适合miRNA-qPCR方法的参考基因。我们使用了一种系统的方法，其中我们利用了其他物种中的类似研究，一些生物学标准，小RNA深度测序的结果，最后，通过qPCR对候选参考基因进行实验验证，以确定最合适的参考基因。Ssa-miR-25- 3 p被确定为最合适的单一参考基因。两个参考基因的最佳组合是ssa-miR-25- 3 p和ssa-miR-455- 5 p。这两个基因在许多不同组织中组成型稳定表达。此外，传染性鲑鱼贫血似乎没有影响他们的表达水平。当应用使用miRNA基因特异性正向引物的简单cybergreen miRNA-PCR方法时，这些基因以高特异性、良好效率扩增，并且qPCR测定显示出良好的线性。我们已经在大西洋鲑鱼中鉴定了用于miRNA-qPCR的合适的参考基因。这些结果为进一步研究该物种的miRNA基因奠定了基础。所鉴定的参考基因是在许多水产养殖物种中成熟序列相同的保守基因。因此，它们也可能适合作为其他硬骨鱼类的参考基因。最后，我们的研究中使用的系统方法成功地确定了合适的参考基因，这表明这可能是一个有用的策略，适用于其他水产养殖物种的类似验证研究。本文的在线版本（doi：10.1186/1756-0500-7-945）包含补充材料，可供授权用户使用。
MicroRNAs (miRNAs) are an abundant class of endogenous small RNA molecules that downregulate gene expression at the post-transcriptional level. They play important roles by regulating genes that control multiple biological processes, and recent years there has been an increased interest in studying miRNA genes and miRNA gene expression. The most common method applied to study gene expression of single genes is quantitative PCR (qPCR). However, before expression of mature miRNAs can be studied robust qPCR methods (miRNA-qPCR) must be developed. This includes identification and validation of suitable reference genes. We are particularly interested in Atlantic salmon (Salmo salar). This is an economically important aquaculture species, but no reference genes dedicated for use in miRNA-qPCR methods has been validated for this species. Our aim was, therefore, to identify suitable reference genes for miRNA-qPCR methods in Salmo salar. We used a systematic approach where we utilized similar studies in other species, some biological criteria, results from deep sequencing of small RNAs and, finally, experimental validation of candidate reference genes by qPCR to identify the most suitable reference genes. Ssa-miR-25-3p was identified as most suitable single reference gene. The best combinations of two reference genes were ssa-miR-25-3p and ssa-miR-455-5p. These two genes were constitutively and stably expressed across many different tissues. Furthermore, infectious salmon anaemia did not seem to affect their expression levels. These genes were amplified with high specificity, good efficiency and the qPCR assays showed a good linearity when applying a simple cybergreen miRNA-PCR method using miRNA gene specific forward primers. We have identified suitable reference genes for miRNA-qPCR in Atlantic salmon. These results will greatly facilitate further studies on miRNA genes in this species. The reference genes identified are conserved genes that are identical in their mature sequence in many aquaculture species. Therefore, they may also be suitable as reference genes in other teleosts. Finally, the systematic approach used in our study successfully identified suitable reference genes, suggesting that this may be a useful strategy to apply in similar validation studies in other aquaculture species. The online version of this article (doi:10.1186/1756-0500-7-945) contains supplementary material, which is available to authorized users.