Chen Yongxin, Gelfond Jonathan A L, McManus Linda M, Shireman Paula K
Department of Surgery, University of Texas Health Science Center, San Antonio, TX 78229, USA.
BMC Genomics. 2009 Aug 28;10:407. doi: 10.1186/1471-2164-10-407.
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).
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.
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.
微小RNA（miRNA）在各种生物学过程中发挥着关键作用。miRNA谱分析是鉴定正常细胞和疾病过程中差异表达miRNA的重要工具。随着通过计算机预测和实验验证发现的miRNA数量不断增加，高通量miRNA表达分析仍面临技术挑战。我们的研究严格评估了一种新型miRNA表达谱分析方法——定量逆转录聚合酶链反应阵列（qPCR阵列）与使用寡核苷酸微芯片（微阵列）进行miRNA检测相比的性能。
通过比较来自相同RNA样本的重复结果，证明了qPCR阵列具有高重现性。miRNA cDNA的预扩增提高了qPCR阵列的灵敏度，并增加了可检测miRNA的数量。此外，预扩增后miRNA的相对表达水平得以保持。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）表明两个检测平台之间存在相当大的变异性。在两种检测中低表达的miRNA中，重复样本之间观察到更高的变异性。最后，与qPCR阵列相比，使用微阵列观察到miRNA差异表达的假阳性率更高。
我们的研究证明了TaqMan qPCR阵列具有高重现性。对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，逆转录反应不会在结果中引入显著差异。cDNA预扩增的使用提高了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miRNA表达估计中不涉及任何系统偏差。微阵列和qPCR阵列之间的比较表明qPCR阵列具有更高的灵敏度和特异性。
