Validation of SplintQuant using fluorescence fragment analysis. (A) Schematic of SplintQuant-FAM. Donor is 5′ phosphorylated and 3′ 6-FAM-labeled (orange sun). Fragment analysis in FAM channel, producing three distinct products differing in size and elution time. (I) Unmodified donor, (II) adenylated donor, and (III) the ligated product (in gray dotted box). (B) Validation of SplintQuant, using 6-FAM labeled donor DNA oligonucleotide. (Left) Oligonucleotide pair for circSMARCA5 (targeting exons 16–15); (Right) Oligonucleotide pair for linear SMARCA5 RNA (targeting exons 16–17). Ligated product within gray dotted box, with representative traces listing color-coded reaction conditions.
