Chromogenic in situ hybridization methods for microRNA biomarker monitoring of drug safety and efficacy

Abstract

Disease research and treatment development have turned to the impact and utility of microRNA. The dynamic and highly specific expression of these molecular regulators can be used to predict and monitor disease progression as well as therapeutic treatment efficacy and safety, thus aiding decisions in patient care. In situ hybridization (ISH) of biopsy material has become a routine clinical pathology procedure for monitoring gene structure, expression, and sample characterization. For ribonucleic acid (RNA), determining cell source and level of expression of these biomarkers gives insight into the cellular function and physiopathology. Identification and monitoring of microRNA biomarkers are made possible through locked nucleic acid (LNA)™-based detection probes. LNA™ enhances the sensitivity and specificity of target binding, most profoundly so for the short, highly similar, microRNA sequences. We present a robust 1-day ISH protocol for formalin-fixed, paraffin-embedded (FFPE) tissue sections based on microRNA-specific LNA™ detection probes which can be labeled with digoxigenin (DIG) or 6-carboxyfluorescein (FAM) and detected through enzyme-linked specific antibodies that catalyze substrates into deposited chromogen products at the target RNA site. The variety of haptens and detection reagents in combination with LNA™ chemistry offer flexibility and ease to multiple target assessment of therapeutic response.