Application of housekeeping npcRNAs for quantitative expression analysis of human transcriptome by real-time PCR
Application of housekeeping npcRNAs for quantitative expression analysis of human transcriptome by real-time PCR
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DOI：
10.1261/rna.1755810
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发表时间：
2010-02-01
期刊：
影响因子：
4.5
通讯作者：
Konthur, Zoltan
中科院分区：
文献类型：
--
作者：
Galiveti, Chenna R.;Rozhdestvensky, Timofey S.;Konthur, Zoltan
近年来，高通量基因表达分析的进步导致了大量非蛋白质编码RNA(NpcRNA)分子的发现。它们形成了一类丰富的未翻译的RNA，已证明在细胞中的不同生化途径中发挥着关键作用。逆转录-实时聚合酶链式反应(RT-qPCR)是一种测量微量物质中RNA丰度和基因表达水平的有效工具。尽管它很敏感，但缺乏准确数据分析所需的适当内部控制是其在npcRNA研究中应用的限制因素。常见的内部对照是编码蛋白质的参考基因，也被称为“管家”基因(HKGs)。然而，据报道，它们的表达水平在不同的组织和不同的实验条件下有所不同。此外，由于生物发生的差异，将HKGs作为npcRNA表达分析的参考是值得怀疑的。为了解决npcRNA分析中最佳RT-qPCR规格子的问题，我们在20个不同的人体组织中对18个npcRNA和4个常见的HKG进行了系统的评估。为了以最小的表达方差确定最合适的内部控制，采用了geNORM、NormFinder、BestKeeper和比较Delta C-Q法四种评价策略。我们的数据有力地表明，五个npcRNA，我们称之为管家RNA(HKR)，在20个不同的人类组织中表现出明显高于普通HKGs的结构性表达水平。已确定的HKR是人类转录组分析中RT-qPCR数据标准化的理想候选者，也可能被用作参考基因，而不考虑被研究基因的性质。
In recent years the improvements in high-throughput gene expression analysis have led to the discovery of numerous non-protein-coding RNA (npcRNA) molecules. They form an abundant class of untranslated RNAs that have shown to play a crucial role in different biochemical pathways in the cell. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is an efficient tool to measure RNA abundance and gene expression levels in tiny amounts of material. Despite its sensitivity, the lack of appropriate internal controls necessary for accurate data analysis is a limiting factor for its application in npcRNA research. Common internal controls applied are protein-coding reference genes, also termed "housekeeping" genes (HKGs). However, their expression levels reportedly vary among tissues and different experimental conditions. Moreover, application of HKGs as reference in npcRNA expression analyses is questionable, due to the differences in biogenesis. To address the issue of optimal RT-qPCR normalizers in npcRNA analysis, we performed a systematic evaluation of 18 npcRNAs along with four common HKGs in 20 different human tissues. To determine the most suitable internal control with least expression variance, four evaluation strategies, geNORM, NormFinder, BestKeeper, and the comparative delta C-q method, were applied. Our data strongly suggest that five npcRNAs, which we term housekeeping RNAs (HKRs), exhibit significantly better constitutive expression levels in 20 different human tissues than common HKGs. Determined HKRs are ideal candidates for RT-qPCR data normalization in human transcriptome analysis, and might also be used as reference genes irrespective of the nature of the genes under investigation.