MASQ: A multiplex qPCR platform for quantitative alternative splicing analysis in clinical specimens

Alternative splicing quantification is critical for understanding disease mechanisms and developing precision medicine approaches. Conventional gel-based methods suffer from poor resolution and limited quantitative precision, especially for similar-sized exons. Here, we present multiplex alternative splicing quantification (MASQ), a dual TaqMan probe qPCR platform enabling precise quantification of alternative splicing events. MASQ employs FAM-labeled probes targeting the alternative exon and HEX-labeled probes for constitutive exon normalization, providing internal controls and eliminating variability. Using PBRM1 exon 27 as a model system, where inclusion promotes immune checkpoint resistance through enhanced PBAF complex recruitment to the PD-L1 promoter, we demonstrate superior analytical performance. MASQ exhibits exceptional linearity (R ² > 0.98), high precision (intra-assay CV < 9%), and femtogram-level sensitivity (LOD 195 fg). CRISPR-engineered cellular validation confirmed probe specificity, while analysis of uterine corpus endometrial carcinoma specimens revealed significantly elevated exon27 inclusion in cancer tissues (61.2% ± 14.5%) compared to normal endometrium (12.7% ± 4.0%, p < 0.01). Platform generalizability was demonstrated through adaptation to HTRA2 exon 7 quantification and monitoring splice-switching oligonucleotide effects (44.6% reduction at 24 h, p < 0.0001). MASQ represents a broadly applicable platform for alternative splicing analysis, enabling applications including biomarker discovery, diagnostic development, and monitoring.