Amplification protocols introduce systematic but reproducible errors into gene expression studies.

The desire to perform microarray experiments with small starting amounts of RNA has led to the development of a variety of protocols for preparing and amplifying mRNA. This has consequences not only for the standardization of experimental design, but also for reproducibility and comparability between experiments. Here we investigate the differences between the Affymetrix standard and small sample protocols and address the data analysis issues that arise when comparing samples and experiments that have been processed in different ways. We show that data generated on the same platform using different protocols are not directly comparable. Further, protocols introduce systematic biases that can be largely accounted for by using the correct data analysis techniques.