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PMID: 19715577 Published · epublish English Comparative Study Journal Article Research Support, N.I.H., Extramural Research Support, U.S. Gov't, Non-P.H.S.

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

BMC genomics ·Vol. 10 ·2009-08-28 ·Pages 407

Chen Y, Gelfond JA, McManus LM, Shireman PK

Abstract

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.

MeSH Terms
Animals Cell Line Gene Expression Profiling/methods Mice MicroRNAs/genetics Oligonucleotide Array Sequence Analysis/methods Reproducibility of Results Reverse Transcriptase Polymerase Chain Reaction/methods Sensitivity and Specificity Sequence Analysis, RNA
Chemicals
MicroRNAs
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Chen Yongxin
Department of Surgery, University of Texas Health Science Center, San Antonio, TX 78229, USA. cheny4@uthscsa.edu
Gelfond Jonathan A L
McManus Linda M
Shireman Paula K
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Article Info
Journal
BMC genomics
Abbr.
BMC Genomics
ISSN
1471-2164
DOI
10.1186/1471-2164-10-407
Email
cheny4@uthscsa.edu
Published
2009-08-28
Epub
2009-00-28
Pages
407
Language
English
Region
England
NLM ID
100965258
PMCID
PMC2753550
Subset
IM
Grants
NHLBI NIH HHS · T32HL07446 · United States
NHLBI NIH HHS · T32 HL007446 · United States
NCATS NIH HHS · UL1 TR000149 · United States
NHLBI NIH HHS · R01HL074236 · United States
NCATS NIH HHS · KL2 TR000118 · United States
NCRR NIH HHS · UL1 RR025767 · United States
NCRR NIH HHS · KL2 RR025766 · United States
NHLBI NIH HHS · R01 HL074236 · United States
External Links
PubMed source DOI: 10.1186/1471-2164-10-407 PMC Full Text View journal details
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