Single-molecule quantification of BCR-ABL1 fusion transcripts in a single-tube workflow using PCR-based RNA counting
Accurate and streamlined methods for quantifying BCR–ABL1 RNA transcripts are essential for monitoring treatment response in chronic myeloid leukemia (CML).
Here, we present a single-tube workflow for the detection of the three major BCR-ABL1 isoforms (e14a2, e13a2, and e1a2) together with the reference ABL1 gene, integrating compartmentalization, reverse transcription (RT), PCR, and direct counting.
Using the one-step Countable RT-PCR platform, up to one million RNA molecules were accurately quantified with single-molecule resolution. Even in the presence of high background ABL1, different levels of BCR-ABL1 variants were reliably detected. Our results closely matched expected %IS values from the reference panel, with successful detection down to 0.0032% BCR-ABL1 and low %CV across all levels.
Our ability to directly count RNA molecules across a broad dynamic range makes the platform well-suited for minimal residual disease (MRD) monitoring. By combining sensitivity, specificity, and workflow efficiency in a single-tube format, our approach offers a scalable solution for RNA-based diagnostics.
