Development and Optimization of an RT-qPCR Assay for Determining miRNA-1202 Expression Levels in Human Blood Samples

Abstract

Objectives: To develop a protocol and optimize RT-qPCR reaction conditions for determining the expression levels of miRNA-1202 in human peripheral venous blood. Materials and Methods: Peripheral blood samples were collected from 10 healthy volunteers, strictly adhering to the requirements for biological sample collection and preservation. Quantitative PCR reaction conditions were optimized, including primer annealing temperature, primer concentration, and template concentration. Optimization efficiency was evaluated based on the analysis of amplification curves and melting curves. Using the optimized parameters, the RT-qPCR protocol was established and finalized. The relative expression level of miRNA-1202 was calculated using the Livak method (2−ΔΔCt), with miRNA-16 serving as the endogenous control. Results: The optimal reaction conditions included a primer concentration of 125 nM, an annealing temperature of 65°C, and a template concentration of 10 ng/µL. The study successfully established an RT-qPCR assay for determining miRNA-1202 expression levels utilizing these optimal conditions.