Jeroen van de Peppel, Patrick Kemmeren, Marijana Radonjic, Harm van Bakel, Dik van Leenen and Frank C.P. Holstege
Genomics Laboratory, Department of Biomedical Genetics, UMC Utrecht
Expression profiling using DNA microarrays has become a universal tool with a host of applications that benefit from accurate determination of differential gene expression (refs). Current microarray analysis assumes that most transcript levels are invariant between samples or that changes are balanced. These assumptions are made to allow normalization on genes. Normalization counters non-biological variation between samples, such as differences in labeled material, dye-biases etc. (refs). Normalization on genes results in changes calculated relative to the behavior of the majority of transcripts. If global mRNA shifts occur, such relative changes will not reflect the absolute changes at the cellular level. We have investigated the occurrence of global changes in microarray experiments using external RNA controls. External control normalization accurately determined mRNA changes in experiments that artificially mimic global mRNA shifts. When applied to yeast stationary phase and human heat-shock, significant mRNA changes for the vast majority of genes were revealed. Even with a serum-starvation experiment exhibiting a modest global change, normalization with external controls had a significant impact on the number of transcripts determined to be differentially expressed. These results suggest that global mRNA changes occur more frequently than previously thought and demonstrate that monitoring such effects is important for accurate determination of gene expression changes.