Systematic Evaluation of Three microRNA Profiling Platforms: Microarray, Beads Array, and Quantitative Real-Time PCR Array

BackgroundA number of gene-profiling methodologies have been applied to microRNA research. The diversity of the platforms and analytical methods makes the comparison and integration of cross-platform microRNA profiling data challenging. In this study, we systematically analyze three representative microRNA profiling platforms: Locked Nucleic Acid (LNA) microarray, beads array, and TaqMan quantitative real-time PCR Low Density Array (TLDA). Methodology/Principal FindingsThe microRNA profiles of 40 human osteosarcoma xenograft samples were generated by LNA array, beads array, and TLDA. Results show that each of the three platforms perform similarly regarding intra-platform reproducibility or reproducibility of data within one platform while LNA array and TLDA had the best inter-platform reproducibility or reproducibility of data across platforms. The endogenous controls/probes contained in each platform have been observed for their stability under different treatments/environments; those included in TLDA have the best performance with minimal coefficients of variation. Importantly, we identify that the proper selection of normalization methods is critical for improving the inter-platform reproducibility, which is evidenced by the application of two non-linear normalization methods (loess and quantile) that substantially elevated the sensitivity and specificity of the statistical data assessment. ConclusionsEach platform is relatively stable in terms of its own microRNA profiling intra-reproducibility; however, the inter-platform reproducibility among different platforms is low. More microRNA specific normalization methods are in demand for cross-platform microRNA microarray data integration and comparison, which will improve the reproducibility and consistency between platforms.

研究背景：多种基因分析方法已被应用于微小核糖核酸（microRNA）研究领域。由于平台类型与分析手段的多样性，跨平台微小核糖核酸表达谱数据的比较与整合始终是颇具挑战性的工作。本研究系统分析了三类具有代表性的微小核糖核酸表达谱分析平台：锁核酸（Locked Nucleic Acid, LNA）微阵列、微球芯片以及TaqMan定量实时聚合酶链反应低密度阵列（TaqMan quantitative real-time PCR Low Density Array, TLDA）。
研究方法与主要结果：本研究通过LNA微阵列、微球芯片及TLDA三个平台，完成了40例人骨肉瘤异种移植样本的微小RNA表达谱构建。结果显示，三类平台在平台内重现性（即单一平台内部的数据一致性）上表现相当，其中LNA微阵列与TLDA的平台间重现性（即跨平台的数据一致性）最优。针对各平台内置的内参对照/探针在不同处理条件与环境下的稳定性，本研究开展了观测评估，结果表明TLDA内置的内参表现最佳，变异系数最小。尤为重要的是，本研究证实恰当选择标准化方法对提升平台间重现性至关重要：两种非线性标准化方法（局部加权回归散点平滑法loess与分位数标准化quantile）的应用，显著提升了统计数据评估的灵敏度与特异性。
结论：各平台在自身的微小RNA表达谱分析平台内重现性上均相对稳定，但不同平台间的跨平台重现性仍处于较低水平。当前亟需更多针对微小RNA的特异性标准化方法，以实现跨平台微小RNA微阵列数据的整合与比较，进而改善不同平台间的数据重现性与一致性。