SplintQuant with Universal qPCR accurately quantifies circRNAs in total RNA. (A) Schematic of SplintQuant. Donor is 5′ phosphorylated and both acceptor and donor are tagged at their termini with M13R and M13F primers, respectively. Following Splint ligation, products are used as templates for qPCR with M13F/M13R primers. (B) Dilution series of circSMARCA5 and linear SMARCA5 showing high degree of correlation over 8-log dynamic range. Slope ± SEM calculated using linear regression and r2 value shown. (C) Calculation of transcript abundance by SplintQuant using 2−ΔΔCt method for six synthetic in vitro transcribed RNAs in either their linear form (unfilled boxes) or circular RNA form (filled boxes). n = 3 biological replicates, in technical triplicate. Linear RNA (600 nt) set as reference expression level of 1. Mean ± SD. (D) Abundance of circSMARCA5, SMARCA5 linear, circDOCK1, and DOCK1 linear RNA in HMLE cellular RNA using SplintQuant. Comparison of normalization with GAPDH SplintQuant (gray filled columns) or GAPDH qRT-PCR (gray checkered columns). Mean ± SD. n = 3 biological replicates, in technical duplicate. Expression shown relative to circSMARCA5, as a value of 1. (E) Abundance of MLL, AF4, and MLL/AF4 transcripts in three human cell leukaemic lines (MV4; 11, MOLM-13, and HL-60). Mean ± SD. n = 3 biological replicates, in technical duplicate; n.d., not detected. (*) P < 0.05 (Student's t-test).
