FROM BIOFLUID TO BIOMARKER: OPTIMIZING MICRORNA PROFILING BY RT-QPCR

in plasma and serum, even in the presence of many RNases. The analytical signal is, however, a composite result of blood collection, clotting or anticoagulation, platelet and erythrocyte carryover, storage, RNA recovery, reverse-transcription chemistry, amplification efficiency, and normalisation. This comprehensive evaluation assesses the whole analytical process from the management of whole blood/plasma/serum to RNA extraction, reverse transcription, and quantitative PCR. Contemporary data supports the use of matrix-specific standard operating procedures, clear quality control for haemolysis and platelet contamination, validated low-input extraction with stage-specific spike-ins, assay-specific reverse transcription validation, and normalisation utilising validated endogenous controls, global mean methods, or absolute quantification when suitable. U6 and RNU48 must not be regarded as universal extracellular reference standards. Digital PCR enhances precise quantification at minimal copy numbers; yet, it does not eradicate pre-analytical or reverse transcription biases. MIQE 2.0 (2025) enhances the reporting standards for assay design, efficacy, dynamic range, limits of detection/quantification, controls, and normalisation. Clinical translation requires a finalised Standard Operating Procedure, calibration particular to the matrix, orthogonal validation, and prospective external validation.