TRANSCRIPTOME 2002: From Functional Genomics to Systems
The Use of Representative Gene Fragments (Rgss) for Less Populated, Highly Replicated Microarrays
Helena B. Samaia, André C. Zaiats, Otávia O. Caballero, Ricardo R Brentani, Sandro J. de Souza and Andrew J.G. Simpson, Ludwig Institute of Cancer Research, São Paulo, BRAZIL
The possibility of extracting the expression profile of thousand of genes in a single experiment makes the use of microarray technology very promissing. The extraction of reliable information from the large data sets generated is critical. Processes including probe selection, spotting, hybridization, image processing and normalization are the major causes of data variability and have been estensively discussed in the literature. However, the potential for hybridization cross-reactivity, the length of the immobilized fragments and the use of replicate spots have been less often reported in these studies. We developed an array using transcript fragments that have been carefully designed so as not to exhibit cross reactivity and that have similar lengths, GC content and position within the transcript (RGSs). They were produced by RT-PCR using primers designed by an in-house computer algorithm. Our current aproach to the statistical analysis of our data also involves working with the variation between replicate spots on the array. We show evidence that hybridization intensity is dependent on the length and the position within the transcript from wich the RGS is derived, that the use of RGS avoids cross-hybridization and that the use of multiple spotting is essential for a reliable interpretation of the data.
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