Application of gene specific mRNA level determinations in individual cells using flow cytometry-based PrimeFlow™ in immunotoxicology

摘要

Determining changes in gene expression by measuring mRNA levels is an important capability in biological research. Real-Time Quantitative PCR (RT-qPCR) is the most ubiquitous technique for measuring changes in mRNA transcript levels, but heterogeneity of cell populations and low cell number are serious technical limitations. Recent advances in flow cytometric analytical techniques have enabled the quantification of mRNA levels in individual cells. Here, we present examples demonstrating the strength and challenges of concurrently measuring mRNA using PrimeFlow™ with other endpoints in immunotoxicological studies. Specifically, we demonstrate how measuring gene specific mRNA levels on a per cell basis was used to study:1) markers of activation and differentiation; 2) cell signaling by measuring intracellular proteins in mature and developing cell types; and 3) a cell type that constitutes a minor population in peripheral blood. We also discuss cell type-specific modifications to the parent technique, which facilitated optimal performance in these cells. While the examples provided are focused on immunotoxicological questions and endpoints, this new strategy can be applied to a wide variety of toxicological research problems.