In PLoS One this week, investigators at Helicos BioSciences say that for RNA-seq analysis, "it is essential to understand how technical variation impacts the quality and interpretability of results, how potential errors could be introduced by the protocol, how the source of RNA affects transcript detection, and how all of these variations can impact the conclusions drawn." By running a variety of human RNA samples through fragmentation, fractionation, cDNA synthesis, and single- versus multiple-tag counting protocols, the Helicos team assessed the advantages and disadvantages of