Methodology article

Reproducibility of quantitative RT-PCR array in miRNA expression profiling and comparison with microarray analysis

Yongxin Chen^{* 1,2} , Jonathan AL Gelfond^{* 3} , Linda M McManus^4,5,7 and Paula K Shireman^1,2,6,7

¹Department of Surgery, University of Texas Health Science Center, San Antonio, TX 78229, USA

²Department of Surgery, South Texas Veterans Health Care System, San Antonio, TX 78229, USA

³Department of Epidemiology and Biostatistics, University of Texas Health Science Center, San Antonio, TX 78229, USA

⁴Department of Pathology, University of Texas Health Science Center, San Antonio, TX 78229, USA

⁵Department of Periodontics, University of Texas Health Science Center, San Antonio, TX 78229, USA

⁶Department of Medicine, University of Texas Health Science Center, San Antonio, TX 78229, USA

⁷Sam and Ann Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center, San Antonio, TX 78229, USA

author email corresponding author email* Contributed equally

BMC Genomics 2009, 10:407doi:10.1186/1471-2164-10-407

Published:	28 August 2009

Abstract

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.