Setting up a microarray platform

MARIANO SALIBE; MARIANO ALVAREZ; DANIELA CELI; CINTIA SERRA; FERNANDO PITOSSI; OSVALDO PODHAJCER

Pinamar, Argentina

Congreso; 10th Congress of the Panamerican Association for Biochemistry and Molecular Biology; 2005

In the last years Microarray technology has become in a powerfull tool for the study of gene expression. To date exists numerous of alternative protocols for every step, all of which may have enormous influence in the final results. Our objective was to study in detail different alternatives for each of these steps, in order to obtain the highest quality results. Here we describe which are the most important variables affecting Microarray printing, target sequences labeling, ARN amplification, hybridization, scanning, and data analysis.  The hybridized Microarray slides were scanned and digitalized using a confocal scanner, data was normalized using different methods, including one developed at our lab.  The experimental designs were analyzed using different statistics methods, like t-student, SAM, ANOVA and Limma and the presence of molecular signatures were evaluated using different clustering algorithms. Conclusions: Highest quality spots were obtained using DMSO 50% as printing buffer on an Aminosilane coated slide. The dynamic range of the scanner is maximized by successive scans varing the sensitivity of the scanner. The most efficient normalization method was the 3D, developed at our lab. A Dye swap design is critical for obtaining high quality results