Technical Analysis of cDNA Microarrays

Cinda P Scott,Jeff Vanwye,M Danielle Mcdonald,Douglas L Crawford,Grzegorz Kudla

doi:10.1371/journal.pone.0004486

Abstract

BackgroundThere 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.Methodology/Principal FindingsIn 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.ConclusionThere 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.

Highlights

Microarrays simultaneously quantify several hundred to thousands of genes on a single glass slide and their use has greatly expanded the breadth of quantified gene expression [1,2,3,4,5,6,7,8,9,10]
Of Cy dye) represents 0.16, 16, 56, 106, 506 the concentration of RNA typically used on F. heteroclitus cDNA microarrays [9,26,28,29,30] (Table 2, MIAME GSE12858)
Among the 329 metabolic genes on the array, 212 of these genes met our criteria of being less than 95% of the photomultiplier tube (PMT) saturation and more than two standard deviations above the negative controls (Ctenophore cDNA with no similarity to vertebrate genes)

Summary

Introduction

Microarrays simultaneously quantify several hundred to thousands of genes on a single glass slide and their use has greatly expanded the breadth of quantified gene expression [1,2,3,4,5,6,7,8,9,10]. For the teleost fish Fundulus heteroclitus variation among individuals in mRNA expression is extensive: .60% of genes have significant differences in expression among individuals within a population [1,9,16,17]. Many of these differences in gene expression are associated with variation in cardiac metabolism [9]. 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

Methods

Results

Discussion

Conclusion