OM-sqPCR enables one-Step multiplex quantification of circulating small-RNA subclasses for colorectal cancer classification

Here, we develop OM-sqPCR, a closed-tube, one-step multiplex RT-qPCR platform that simultaneously quantifies microRNAs, transfer RNA–derived sRNAs (tsRNAs), and ribosomal RNA–derived sRNAs (rsRNAs). By integrating optimized reaction chemistry for short RNA templates with computational primer–probe design, OM-sqPCR enables coordinated amplification of multiple sRNA subclasses in a single reaction.

Applied to a multicentre cohort of 651 participants, OM-sqPCR identified a four-sRNA classifier that discriminated colorectal cancer from controls with an area under the ROC curve of 0.97, 85.9% sensitivity, and 93.1% specificity, with sustained performance in early-stage disease. The platform also supported measurement of previously reported sRNA panels for renal and thyroid cancer. These findings establish OM-sqPCR as a practical approach for multiplex circulating sRNA analysis and clinical biomarker evaluation.