Digital PCR-Based Gene Expression Analysis Using a Highly Multiplexed Assay with Universal Detection Probes to Study Induced Pluripotent Stem Cell Differentiation into Cranial Neural Crest Cells.

Induced pluripotent stem cells (iPSCs) have the potential to differentiate into any cell type, offering a valuable tool for research in developmental biology, regenerative medicine, and disease modeling. In this study, iPSCs were differentiated into cranial neural crest cells (CNCCs) over a 14-day period. RNA was extracted from these cells at day 0 (iPSCs), day 7, and day 14 to evaluate successful differentiation through the expression analysis of pluripotency and CNCC marker genes.A key focus was the conversion of existing qPCR assays into multiplexed RT-dPCR assays utilizing universal detection probes for precise gene expression analysis during the differentiation of induced pluripotent stem cells (iPSCs) into cranial neural crest cells (CNCCs). We aimed to leverage the superior precision, sensitivity, and multiplexing-degree of dPCR, particularly in quantifying low-abundance targets. We conducted a comparative analysis of the temporal expression patterns of crucial marker genes using both qPCR and dPCR.Our experiments revealed that the four five-plex dPCR assays could successfully detect and quantify the pluripotency and CNCC marker genes and evaluate CNCC differentiation. We observed the expected downregulation of pluripotency genes during differentiation. Conversely, the upregulation of CNCC markers validates the successful differentiation process. In conclusion, SYBR Green I gene expression qPCR assays can be readily converted into multiplex dPCR assays using universal detection probes.Overall, this work underscores the potential of dPCR as a valuable tool for molecular profiling in stem cell research, offering robust, precise, and efficient gene expression analysis. The findings suggest that while qPCR remains a reliable method for routine applications, dPCR provides particular advantages for high-precision, low-sample input studies, expanding the analytical toolbox for stem cell differentiation and gene expression research.