RNA spike-ins are samples of RNA at known concentrations that can be used as gold standards in experimental design and during downstream analyses for absolute quantification and detection of genome-wide effects.
* Absolute quantification: Absolute quantification of gene expression is not possible with most RNA-Seq experiments, which quantify expression relative to all transcripts. It is possible by performing RNA-Seq with spike-ins, samples of RNA at known concentrations. After sequencing, read counts of spike-in sequences are used to determine the relationship between each genes read counts and absolute quantities of biological fragments. In one example, this technique was used in Xenopus tropicalis' embryos to determine transcription kinetics.
* Detection of genome-wide effects: Changes in global regulators including chromatin remodelers, transcription factors (e.g., MYC), acetyltransferase complexes, and nucleosome positioning are not congruent with normalization assumptions and spike-in controls can offer precise interpretation.