RESSq-PCR assay primer design and RNA editing fingerprint validation in stable human ADAR1-overexpressing cells. (A,B) Primer design strategy showing RNA editing site-specific qRT-PCR (RESSq-PCR) primer design strategy (1) to selectively detect wild-type (A) or edited (G/I) bases using the Tetra-primer amplification refractory mutation system (ARMS) principles (A). Adaptation of the RESSq-PCR primer design strategy (2) for positions that are not compatible with the Tetra-primer ARMS method due to significant differences in GC content directly upstream and downstream or the edited nucleotide position (B). FW = forward primer, Rev = reverse primer, Pos = positive control flanking primers. (C) RESSq-PCR analysis of MDM2, APOBEC3D, GLI1 and AZIN1 RNA recoding in stably-transduced K562-ADAR1 cells compared with K562 wt, K562-ORF and K562-ADAR1m lines (n = 2-4 per site). *p < 0.05 by unpaired, two-tailed Student’s t-test. (D) qRT-PCR analysis of MDM2, APOBEC3D, GLI1 and AZIN1 relative transcript expression using primers flanking each editing site in wild-type (wt) K562 (n = 5), K562-ORF (n = 5), K562-ADAR1 (n = 5) and K562-ADAR1m (n = 3) cDNA. For calculation of transcript control levels, Ct values were normalized to qRT-PCR Ct values using human-specific primers against HPRT.