Evaluation of reference-based two-color methods for measurement of gene expression ratios using spotted cDNA microarraysReportar como inadecuado




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BMC Genomics

, 7:35

First Online: 24 February 2006Received: 26 September 2005Accepted: 24 February 2006

Abstract

BackgroundSpotted cDNA microarrays generally employ co-hybridization of fluorescently-labeled RNA targets to produce gene expression ratios for subsequent analysis. Direct comparison of two RNA samples in the same microarray provides the highest level of accuracy; however, due to the number of combinatorial pair-wise comparisons, the direct method is impractical for studies including large number of individual samples e.g., tumor classification studies. For such studies, indirect comparisons using a common reference standard have been the preferred method. Here we evaluated the precision and accuracy of reconstructed ratios from three indirect methods relative to ratios obtained from direct hybridizations, herein considered as the gold-standard.

ResultsWe performed hybridizations using a fixed amount of Cy3-labeled reference oligonucleotide RefOligo against distinct Cy5-labeled targets from prostate, breast and kidney tumor samples. Reconstructed ratios between all tissue pairs were derived from ratios between each tissue sample and RefOligo. Reconstructed ratios were compared to i ratios obtained in parallel from direct pair-wise hybridizations of tissue samples, and to ii reconstructed ratios derived from hybridization of each tissue against a reference RNA pool RefPool. To evaluate the effect of the external references, reconstructed ratios were also calculated directly from intensity values of single-channel One-Color measurements derived from tissue sample data collected in the RefOligo experiments. We show that the average coefficient of variation of ratios between intra- and inter-slide replicates derived from RefOligo, RefPool and One-Color were similar and 2 to 4-fold higher than ratios obtained in direct hybridizations. Correlation coefficients calculated for all three tissue comparisons were also similar. In addition, the performance of all indirect methods in terms of their robustness to identify genes deemed as differentially expressed based on direct hybridizations, as well as false-positive and false-negative rates, were found to be comparable.

ConclusionRefOligo produces ratios as precise and accurate as ratios reconstructed from a RNA pool, thus representing a reliable alternative in reference-based hybridization experiments. In addition, One-Color measurements alone can reconstruct expression ratios without loss in precision or accuracy. We conclude that both methods are adequate options in large-scale projects where the amount of a common reference RNA pool is usually restrictive.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2164-7-35 contains supplementary material, which is available to authorized users.

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Autor: Bernardo R Peixoto - Ricardo ZN Vêncio - Camila M Egidio - Luisa Mota-Vieira - Sergio Verjovski-Almeida - Eduardo M Rei

Fuente: https://link.springer.com/article/10.1186/1471-2164-7-35







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