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

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.

Development of a daphnia magna DNA microarray for evaluating the toxicity of environmental chemicals

“…Our results also indicate that it is possible to obtain gene expression profiles using one-color evaluation, based on direct measurement of fluorescent intensities. The advantages of single-channel measurement also have been reported by other groups [16]. From a toxicogenomic point of view, one-color evaluation of DNA microarray data is an ideal method, potentially allowing one to easily obtain multiple data points, such as different chemicals, doses, and times of exposure.…”

“…In this study, we used the RNA standard only to confirm the reproducibility of the glass array, and we did not calculate fluorescent ratios of Cy5 and Cy3. Instead, fluorescent intensities of each spot on the microarray were measured directly [16].…”

Genome mapping and expression analyses of human intronic noncoding RNAs reveal tissue-specific patterns and enrichment in genes related to regulation of transcription

“…Data were extracted from the images with ArrayVision 8.0 (Imaging Research Inc., GE Healthcare, Piscataway, NJ, USA). Cy5- and Cy3-derived intensity data from the same sample were corrected for intensity-dependent dye biases [66] using a Lowess function implemented in the R package [67]. The different experiments with human tissues were normalized by the 40% trimmed mean intensity of all the spots in each slide that were above the mean plus 2 SD intensity of 1,198 negative controls.…”

Análise global da expressão de RNAs não codificadores no sistema imunológico humano na senescência e sepse

“…Um desenho experimental frequentemente utilizado envolve o uso de dois marcadores fluorescentes, que se distinguem pelo comprimento de onda da luz emitida. Ao marcar duas amostras diferentes com cada um dos marcadores e hibridizalas simultaneamente em um mesmo microarranjo, pode-se aumentar significativamente a precisão com que se compara expressão de RNA das amostras (Peixoto, Vencio et al 2006).…”

“…C i é, portanto, a soma da similaridade de todos os genes entre si, normalizados por quanto cada um deles é similar ao gene i (se fosse uma rede sem pesos, onde a ligação entre genes é binária, este seria o número de conexões feitas pelos genes ligados a i), normalizado pela soma das similaridades de i vezes quão mais similar i é que dos outros genes da rede. Em uma analogia usando interações sociais entre pessoas, C i é 1 se todos os seus amigos são amigos entre si, e 0 se nenhum deles se conhece (Zhang and Horvath 2005 (Peixoto, Vencio et al 2006).…”