The influence of RNA integrity,purity and cDNA labelling on glass slide cDNA microarray image quality

The accuracy of gene expression measurements generated using cDNA microarrays is dependent on the quality of the image generated following hybridization of fluorescently labelled cDNA. It is not known how this image is influenced by sample preparation factors which such as RNA quality, cDNA synthesis and labelling efficiency. In this study we used a simple metric based on the ratio of the total feature (F) and background (B) fluorescence, which correlates with the visual assessment of 60 microarray images, to determine the influence of sample preparation on image quality. Results indicate that RNA purity (A260/A280) and integrity (18S:28S ratio) do not strongly influence microarray image quality. cDNA having an nucleotide to dye ratio greater than 100 produced poor microarray images, however, cDNA labelled more efficiently was not a guarantee of a better image. The data also indicate that the array image quality is not improved by loading more cDNA into the hybridization mixture however poor image quality did result from a disproportionate amounts of Cy5 and Cy3 labelled cDNA. This study provides insight into the source of variation in microarray image analysis introduced during sample preparation and will assist in the standardisation of cDNA glass slide microarray protocols.