When RNA goes off script: ensuring transcript fidelity in transgene expression.

Plasmids are the workhorses of molecular biology: fast, flexible, and often taken for granted. We clone, overexpress, tag, and mutate freely, assuming they will faithfully produce RNA transcripts that match the intended DNA sequence. This assumption is rarely tested and often invalidated. Sequences in plasmid backbones, epitope tags, and codon-optimized regions may inadvertently harbor cryptic promoters or splice sites. The resulting unexpected transcripts and proteins, while often undetected, can distort results and propagate false conclusions through papers, grants, and even clinical trials. In this perspective, we highlight published cases where plasmids have distorted results and misled interpretation. We examine the mechanisms and consequences of plasmid-associated expression artifacts and offer practical strategies to minimize them. Finally, we call for a revision of community standards for experiments using transgenes: deposit complete plasmid sequences and verify the resulting transcripts using RNA-seq.