MicroRNA-451a in Colorectal Cancer Detection: Analytical and Preliminary Clinical Validation of a PCR-Free Chemiluminescent Assay

Faecal microRNAs are promising molecular biomarkers for non-invasive colorectal cancer detection because of their stability in stool and disease-associated expression profiles. miR-451a is highly enriched in red blood cells and is therefore linked to erythrocyte-derived material associated with lesion-related bleeding. Here, we developed a direct chemiluminescent Dynamic Chemistry Labelling assay for miR-451a detection in a non-invasive colorectal cancer testing workflow. A chemiluminescent Dynamic Chemistry Labelling assay was developed for direct analysis of clarified stool lysates without RNA extraction, reverse transcription or enzymatic amplification. Analytical performance was evaluated using synthetic canonical miR-451a, abundant miR-451a isomiR variants and red blood cell spike-ins. Clinical performance was assessed in stool samples from 90 colonoscopy-characterised individuals, including 44 colorectal cancer cases and 46 colonoscopy-negative controls. Small RNA sequencing was used as an orthogonal comparison method. Group differences were assessed using non-parametric tests, agreement with sequencing data using Spearman correlation, and diagnostic performance using colonoscopy as the reference standard. The assay achieved a limit of detection of 4.25 pg/mL and a lower limit of quantification of 13 pg/mL. It detected canonical miR-451a and its abundant isomiR variants with sequence specificity. Red blood cell spike-in experiments confirmed detection of erythrocyte-derived miR-451a within the stool matrix, with the lowest quantifiable input corresponding to 1,689 red blood cells/µL. Using a predefined positivity threshold of 26 pg/mL, 36 of 44 colorectal cancer cases were miR-451a-positive and 36 of 46 colonoscopy-negative controls were miR-451a-negative, corresponding to 81.8% sensitivity and 78.3% specificity. Ten colonoscopy-negative controls were miR-451a-positive, whereas eight colorectal cancer cases were below the threshold. Dynamic Chemistry Labelling showed a positive association with small RNA sequencing and detected miR-451a in selected colorectal cancer samples with sequencing non-detection. This study establishes a direct PCR-free strategy for interrogating microRNAs in stool samples. miR-451a provides a sequence-defined molecular readout of erythrocyte-derived material and a CRC-relevant target suitable for stool-based nucleic acid testing. This approach provides a translational route toward future multiplexed faecal microRNA panels for colorectal cancer detection.