The genetic code describes how each nucleotide triplet, or codon, of an mRNA encodes a specific amino acid or stop codon. There are 61 codons that encode 20 amino acids; most amino acids are therefore encoded by two or more synonymous codons. It was shown early on that the choice between synonymous codons influences protein output, but the mechanism was unclear. One possibility was that the speed of translation elongation reflected the supply and demand of tRNAs; however, experimental evidence was scarce because availability of charged tRNAs is difficult to measure owing to their secondary structure and modifications.

Another research group soon reported that codon optimality is also observed in zebrafish, clawed frogs, mice and fruitflies during the maternal-to-zygotic transition, a period when maternal mRNAs are degraded to accommodate the transcriptional activation of the zygotic genome. In addition to calculating CSCs based on mRNA decay rates, Bazzini et al. injected zebrafish and frog embryos with a reporter library and measured the resulting RNA concentrations 8–9 h after injection, in the presence or absence of an inhibitor of reporter translation. This experiment revealed a similar set of optimal and non-optimal codons as the CSCs.