Technical analysis of cDNA microarrays

• Published in: PloS one Vol. 4; no. 2; p. e4486
• Main Authors: Scott, Cinda P, VanWye, Jeff, McDonald, M Danielle, Crawford, Douglas L
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 16.02.2009; Public Library of Science (PLoS)
• Subjects: Animals; Atmospheric sciences; Binding sites; Biological variation; Biotechnology; Blood; Deoxyribonucleic acid; DNA; DNA microarrays; Experiments; Fish; Fisheries; Fluorescence; Fluorescent Dyes - metabolism; Fundulidae - genetics; Fundulus heteroclitus; Gene Expression; Gene Expression Profiling - methods; Genes; Genetics and Genomics/Gene Expression; Genomes; Hybridization; Labeling; Labelling; Laboratories; Marine biology; Messenger RNA; Molecular Biology; Oligonucleotide Array Sequence Analysis - methods; Oncorhynchus mykiss; Quality control; Ribonucleic acid; RNA; RNA - genetics; RNA - metabolism; RNA polymerase; Stochasticity; Stress response; Trout; Variation; Florida; United States > US
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0004486

There is extensive variation in gene expression among individuals within and between populations. Accurate measures of the variation in mRNA expression using microarrays can be confounded by technical variation, which includes variation in RNA isolation procedures, day of hybridization and methods used to amplify and dye label RNA for hybridization. In this manuscript we analyze the relationship between the amount of mRNA and the fluorescent signal from the microarray hybridizations demonstrating that for a wide-range of mRNA concentrations the fluorescent signal is a linear function of the amount of mRNA. Additionally, the separate isolation, labeling or hybridization of RNA does not add significant amounts of variation in microarray measures of gene expression. However, single or double rounds of amplification for labeling do have small but significant affects on 10% of genes, but this source of technical variation is easy to avoid. To examine both technical and stochastic biological variation, mRNA expression was measured from the same five individuals over a six-week time course. There were few, if any, meaningful differences in gene expression among time points. Thus, microarray measures using standard laboratory procedures can be precise and quantitative and are not subject to significant random biological noise.