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

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Alternative splicing quantification is crucial for understanding diseases and developing precision medicine.
• Conventional methods lack resolution and precision for similar-sized exons.

Purpose of the Study:

• To introduce multiplex alternative splicing quantification (MASQ), a dual TaqMan probe qPCR platform.
• To enable precise quantification of alternative splicing events with improved accuracy and sensitivity.

Main Methods:

• MASQ utilizes FAM-labeled probes for alternative exons and HEX-labeled probes for constitutive exon normalization.
• A model system using PBRM1 exon 27 was employed to demonstrate analytical performance.
• CRISPR-engineered cells validated probe specificity.

Main Results:

• MASQ demonstrated high linearity (R ² > 0.98), precision (intra-assay CV < 9%), and sensitivity (LOD 195 fg).
• Significantly elevated PBRM1 exon 27 inclusion was observed in uterine corpus endometrial carcinoma tissues.
• The platform was successfully adapted for HTRA2 exon 7 quantification and splice-switching oligonucleotide monitoring.

Conclusions:

• MASQ is a broadly applicable platform for precise alternative splicing analysis.
• This technology supports biomarker discovery, diagnostic development, and therapeutic monitoring.