Normalization Strategies for miRNA RT-qPCR in Blood-Derived Samples in Obesity Research: Current Practices and Methodological Considerations

Reverse transcription quantitative PCR (RT-qPCR) is widely used to quantify circulating microRNAs, but reliable interpretation depends on appropriate data normalization. In obesity research, selecting reference RNAs for measurements in whole blood, blood cells, plasma, and serum is challenging because of biological heterogeneity and preanalytical variability. This review summarizes normalization strategies used in RT-qPCR studies of blood-derived samples from participants with overweight or obesity. We examine commonly used endogenous reference RNAs, including U6 snRNA and miR-16-5p, and assess the rationale for their selection and the available validation evidence in relation to sample matrix and cohort characteristics. The review evaluates normalization practices and the quality and reporting of reference-RNA validation in obesity-related studies, with particular attention to the biological comparison being made. No candidate, algorithmic ranking, or fixed panel size is considered universally applicable. Candidate selection, the number of references, and the normalization scale should be justified for the intended comparison. The review provides practical guidance for designing and reporting validation of reference small non-coding RNAs and process controls in study cohorts with overweight or obesity, with recommendations qualified by the available evidence.