|
[Abstract] [Full Text] [PDF]
Microarray validation: factors influencing correlation between oligonucleotide microarrays and real-time PCR Jeanine S. Morey1*, James C. Ryan1 and Frances M. Van Dolah1 1 NOAA Marine Biotoxins Program, Center for Coastal Environmental Health and Biomolecular Research. 219 Fort Johnson Rd., Charleston, SC, 29412. USA. Biol. Proced. Online 2006;8:175-193. doi:10.1251/bpo126 Indexing terms: Polymerase Chain Reaction; Microarray Analysis; Gene Expression; Nucleic Acid Amplification Techniques; Reverse Transcriptase Polymerase Chain Reaction; RNA. Abbreviations: AZA, azaspiracid; Ct, cycle threshold; DA, domoic acid; DR, dose response; IP, intraperitoneally; PbTx, brevetoxin; qPCR, quantitative real-time polymerase chain reaction; RT, reverse transcription; TC, time course. |
Quantitative real-time PCR (qPCR) is a commonly used validation tool for confirming gene expression results obtained from microarray analysis; however, microarray and qPCR data often result in disagreement. The current study assesses factors contributing to the correlation between these methods in five separate experiments employing two-color 60-mer oligonucleotide microarrays and qPCR using SYBR green. Overall, significant correlation was observed between microarray and qPCR results (ρ=0.708, p<0.0001, n=277) using these platforms. The contribution of factors including up- vs. down-regulation, spot intensity, ρ-value, fold-change, cycle threshold (Ct), array averaging, tissue type, and tissue preparation was assessed. Filtering of microarray data for measures of quality (fold-change and ρ-value) proves to be the most critical factor, with significant correlations of ρ>0.80 consistently observed when quality scores are applied. |