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

Accurate multiplex quantification of circulating small RNAs (sRNAs) enables comprehensive evaluation of their diagnostic potential. 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 multicenter 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.