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Microarray validation: factors influencing correlation between oligonucleotide microarrays and real-time PCR

Abstract

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 ρτ;0.80 consistently observed when quality scores are applied.

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

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Correspondence to Jeanine S. Morey.

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Morey, J.S., Ryan, J.C. & Van Dolah, F.M. Microarray validation: factors influencing correlation between oligonucleotide microarrays and real-time PCR. Biol. Proced. Online 8, 175–193 (2006). https://doi.org/10.1251/bpo126

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  • DOI: https://doi.org/10.1251/bpo126

Indexing terms

  • Polymerase Chain Reaction
  • Microarray Analysis
  • Gene Expression
  • Nucleic Acid Amplification Techniques
  • Reverse Transcriptase Polymerase Chain Reaction
  • RNA