A comparison of branched DNA and reverse transcriptase quantitative polymerase chain reaction methodologies for...

Messenger RNA (mRNA)-based therapeutics have emerged as a promising modality for various clinical applications, necessitating robust methods for mRNA quantification. This biodistribution study compares the performance of branched DNA and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assays for measuring lipid nanoparticle-encapsulated mRNA. Following intravenous administration of nascent peptide imaging luciferase mRNA (1 mg/kg) to rats, mRNA levels in various tissues and serum were quantified using both assays. Statistical analyses, including Bland-Altman, Deming regression and Passing-Bablok regression, were employed to assess method comparability and reproducibility. The results indicated that mRNA pharmacokinetics measured by branched DNA and RT-qPCR were largely consistent across tissues, with RT-qPCR showing greater reproducibility across multiple laboratories. RT-qPCR also demonstrated a wider dynamic range and higher sensitivity, making it a more versatile option for large-scale studies. Despite some differences in data due to tissue types and timepoints, both methods provided comparable pharmacokinetic profiles for mRNA quantification. This study underscores the importance of selecting an appropriate quantification method based on study requirements and highlights RT-qPCR's adaptability for multisite research, especially for the clinical development of mRNA-based therapeutics.