Reproducibility of quantitative RT-PCR array in miRNA expression profiling and comparison with microarray analysis
Reproducibility of quantitative RT-PCR array in miRNA expression profiling and comparison with microarray analysis
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DOI：
10.1186/1471-2164-10-407
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发表时间：
2009-08-28
期刊：
影响因子：
4.4
通讯作者：
Shireman, Paula K.
中科院分区：
文献类型：
--
作者：
Chen, Yongxin;Gelfond, Jonathan A. L.;Shireman, Paula K.
背景：microRNAs（miRNAs）在多种生物学过程中发挥着重要作用。MiRNA谱是鉴定正常细胞和疾病过程中差异表达的MiRNA的重要工具。随着计算机预测和实验验证，miRNA的数量不断增加，高通量miRNA表达分析仍然存在技术挑战。我们的研究批判性地评估了一种新的miRNA表达谱分析方法，定量RT-PCR阵列（qPCR-array）的性能，与寡核苷酸微芯片（microarray）检测miRNA相比。miRNA cDNA的预扩增提高了qPCR阵列的灵敏度，并增加了可检测的miRNA的数量。此外，在预扩增后，miRNAs的相对表达水平保持不变。当使用相同RNA的不同等分试样比较qPCR-阵列和微阵列的性能时，两种方法之间的低相关性（r = -0.443）表明两种测定平台之间存在相当大的变异性。在两种测定中，在具有低表达的miRNA中观察到重复之间的较高变异。最后，与qPCR-array相比，采用TaqMan qPCR-array检测miRNA差异表达的假阳性率更高。在不同日期进行的不同逆转录反应和qPCR阵列之间的比较表明，逆转录反应未在结果中引入显著变化。cDNA预扩增的使用增加了miRNA检测的灵敏度。尽管低丰度miRNA的预扩增存在变异性，但后者在估计miRNA表达时不涉及任何系统偏倚。微阵列与qPCR-阵列的比较表明，qPCR-阵列具有优越的上级敏感性和特异性。
Background: MicroRNAs (miRNAs) have critical functions in various biological processes. MiRNA profiling is an important tool for the identification of differentially expressed miRNAs in normal cellular and disease processes. A technical challenge remains for high-throughput miRNA expression analysis as the number of miRNAs continues to increase with in silico prediction and experimental verification. Our study critically evaluated the performance of a novel miRNA expression profiling approach, quantitative RT-PCR array (qPCR-array), compared to miRNA detection with oligonucleotide microchip (microarray).Results: High reproducibility with qPCR-array was demonstrated by comparing replicate results from the same RNA sample. Pre-amplification of the miRNA cDNA improved sensitivity of the qPCR-array and increased the number of detectable miRNAs. Furthermore, the relative expression levels of miRNAs were maintained after pre-amplification. When the performance of qPCR-array and microarrays were compared using different aliquots of the same RNA, a low correlation between the two methods (r = -0.443) indicated considerable variability between the two assay platforms. Higher variation between replicates was observed in miRNAs with low expression in both assays. Finally, a higher false positive rate of differential miRNA expression was observed using the microarray compared to the qPCR-array.Conclusion: Our studies demonstrated high reproducibility of TaqMan qPCR-array. Comparison between different reverse transcription reactions and qPCR-arrays performed on different days indicated that reverse transcription reactions did not introduce significant variation in the results. The use of cDNA pre-amplification increased the sensitivity of miRNA detection. Although there was variability associated with pre-amplification in low abundance miRNAs, the latter did not involve any systemic bias in the estimation of miRNA expression. Comparison between microarray and qPCR-array indicated superior sensitivity and specificity of qPCR-array.