Comparison of Amplification Methods to Produce Affymetrix GeneChip(Trademark) Target Material
FEDERAL AVIATION ADMINISTRATION OKLAHOMA CITY OK CIVIL AEROMEDICAL INST
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Whole blood from living subjects is a convenient matrix to use as a source of RNA for microarray experiments with human subjects especially when subject material is collected at a location other than the collaborating site conducting the microarray work. Collection methods for whole blood that include stabilization of the RNA are known but suffer from issues of decreased sensitivity due to the large amount of globin RNA present from reticulocyte lysis. The experiments presented here were designed to test a globin-RNA reduction protocol in conjunction with three different amplification methods. Statistical analysis of the six different protocols, coupled with post-hybridization quality assurance methods, revealed that an amplification protocol that yielded a fragmented biotin-labeled cDNA product resulted in the highest Percent Present calls from the Affymetrix analysis software and the least methodology based variability. Based on these results, this amplification protocol is expected to lead to the greatest sensitivity and accuracy for differential expression testing of the six amplification methods tested.
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