Biotin incorporation measurement. aRNA was digested to nucleosides, then separated by HPLC. The upper panel shows the absorbance profile of the heterocycles at 260 nm. The lower panel shows the absorbance profile at 294 nm. The biotin incorporation rate can be calculated using the area under each peak and the extinction coefficient of each nucleoside. The calculation indicates that a biotin-containing nucleotide is present approximately every 8 bases.