Hardly anyone would run an RNA gel without a ladder, but transcriptomes are mostly sequenced without the use of external standards. The added layer of transcript isoform complexity in eukaryotes as well as incomplete or incorrect gene annotations further challenge RNA-Seq pipelines to correctly calculate and compare gene expression values. Lexogen, a specialized transcriptomics company, addresses this problem by providing Spike-In RNA Variant Control Mixes (SIRVs) to the RNA-Seq community. These controls are processed together with the RNA sample to allow for an evaluation of the RNA-Seq workflow and, in particular, of transcript isoform detection and gene expression quantification. The mixes contain 69 transcript variants that map to 7 human model genes and mirror the native transcriptome complexity by comprehensively representing splicing isoforms, transcription start-site and end-site variants, overlapping transcripts and antisense transcription. Lukas Paul, Head of Services at Lexogen, describes in this webinar how the SIRVs have been used to estimate absolute accuracy and consistency, as well as concordance in gene expression measurements at the level of workflows, experiments, and samples. A “SIRVs dashboard” is introduced that brings together spike-in derived NGS data, annotations and data evaluation in an easily navigable way, and the webinar will highlight how condensed SIRVs data can function as a “RNA-Seq fingerprint” that enables comparisons across experiments, samples and platforms.