Automated target preparation for gene expression analysis eliminates the time-consuming and labor-intensive manual process, which is error prone and diverts scientists from value added activities. Target preparation methods were developed, on the fully integrated ArrayPlex SA� system, based on the field proven Biomek FX�, which streamlined the target preparation procedure allowing up to 96 samples to be processed in less than 36 h. The process is comprised of three functional methods, cDNA synthesis, in vitro transcription, and fragmentation, providing the users with the ability to consolidate runs for optimal use of instrument time and minimize reagent cost. Starting with sets of eight tRNA samples, the cDNA synthesis method synthesizes the first and second strand DNA followed by a cDNA clean-up step using an ultrafiltration plate. The in vitro transcription method then amplifies and biotin-labels the cDNA to cRNA in 6 h at 37�C, and purifies the product using a solid support extraction plate. Finally, the fragmentation method quantifies the cRNA, adjusts the concentration to the recommended 0.625 �g/mL and fragments the cRNA prior to an off-line hybridization. Universal human reference RNA with concentration ranging from 1 to 7.5 �g were prepared on the ArrayPlex SA, compared against a manual method and scanned using Affymetrix human genome U133 Plus 2.0 array GeneChip� cartridges. Nested analysis of variance was then performed to identify sources of variability between the automated and manual methods.