An Optimized Direct Lysis Gene Expression Microplate Assay and Applications for Disease, Differentiation, and Pharmacological Cell-Based Studies

Routine cell culture reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) gene expression analysis is limited in scalability due to minimum sample requirement and multistep isolation procedures. In this study, we aimed to optimize and apply a cost-effective and rapid protocol for directly sampling gene expression data from microplate cell cultures. The optimized protocol involves direct lysis of microplate well population followed by a reduced thermocycler reaction time one-step RT-qPCR assay. In applications for inflammation and stress-induced cell-based models, the direct lysis RT-qPCR microplate assay was utilized to detect IFN1 and PPP1R15A expression by poly(I:C) treated primary fibroblast cultures, IL6 expression by poly(I:C) iPSC-derived astrocytes, and differential PPP1R15A expression by ER-stressed vanishing white-matter disease patient induced pluripotent stem cell (iPSC)-derived astrocytes. In application for neural differentiation medium recipe optimizations, conditions were screened for SYN1 and VGLUT1 in neuronal cultures, and S100B, GFAP and EAAT1 in astrocyte cultures. The protocol provides microplate gene expression results from cell lysate to readout within similar to 35 min, with comparable cost to routine RT-qPCR, and it may be utilized to support laboratory cell-based assays in basic and applied scientific and medical fields of research including stem-cell differentiation, cell physiology, and drug mechanism studies.

Automated summary

This study aimed to optimize and apply a cost-effective protocol for direct sampling of gene expression data from microplate cell cultures, overcoming the limitations of routine RT-qPCR analysis. The optimized protocol involves direct lysis of microplate well population followed by a reduced thermocycler reaction time one-step RT-qPCR assay. The protocol provides quick and cost-comparable microplate gene expression results, which can be utilized in various cell-based applications.