Senior Principal Scientist Pfizer Andover, Massachusetts
qPCR and dPCR are standard methods for quantifying DNA/RNA in evaluating the biodistribution profile of gene therapy products, as recommended by the ICH S12 guideline. However, limited data exist on the comparability of these methods and their respective pros and cons on RNA quantification. We developed and qualified RT-dPCR and RT-qPCR assays using the same primers and probes to quantify IgG heavy and light chain mRNAs in rat serum dosed with LNP-mRNA. Both assays demonstrated accuracy, precision, and specificity. While dPCR and qPCR produced equivalent results for the heavy chain, a systematic difference was observed for the light chain, likely due to variations in RNA reverse transcription efficiency. This presentation will cover challenges in reverse transcription, assay optimization, and validation, addressing analytical challenges to produce essential data for understanding LNP-mRNA biodistribution.
Learning Objectives:
To understand the technology and methodology difference between digital PCR and real-time qPCR platforms, their pros and cons.
To be able to evaluate critical factors in selecting RT-dPCR and RT-qPCR for RNA quantification.
To understand challenges associated with the PCR data and be able to interpret the results.
