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

Abstract

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.

FMRP Levels in Human Peripheral Blood Leukocytes Correlates with Intellectual Disability

“…Although FMR1 unmethylated alleles, even in the full mutation range, [16] are transcribed, they are not efficiently translated into FMRP starting from the upper premutation into the full mutation range [18,48]. Thus, the lower FMRP expression detected in individuals with a full mutation and mosaicism [20,21,41,49] and in individuals carrying a premutation allele [20,21,50] could be responsible for the clinical, cognitive, and behavioral impairment seen in fragile X syndrome and FMR1 associated disorders [14,26].…”

“…The first assay is a quantitative, high throughput, electrochemiluminescence assay [24,25] with a low false detection rate, a 0.07 fmol lower limit of detection (LLOD), and a 0.65 fmol lower limit of quantification (LLOQ). The second assay, based on PrimeFlow TM by ThermoFisher Scientific (Waltham, MA, USA) [26,27], is a flow cytometric assay which simultaneously immunophenotypes PMBCs and measures the relative amounts of FMR1 and FMRP.…”

Bioanalysis in the Age of New Drug Modalities

“…It requires a large number of target cells to extract sufficient mRNA for quantifying the gene expression. Additionally, qPCR is limited in its ability to quantify gene expression using heterogeneous cell preparations and to concurrently measure mRNA and protein ( 66 ). Flow cytometry for the measurement of mRNA and protein is an alternative in characterizing the biodistribution of GTs ( 67 ), though its sensitivity is not comparable to that of qPCR.…”

Single‐Cell Analysis of Cytokine mRNA and Protein Expression by Flow Cytometry

“…Reagent optimization and/or omission may be necessary based on the cell type or intracellular proteins being tested to maintain the sensitivity of target mRNA detection; this is outlined in Henriquez et al. (2017). Tissue and cell types that have been associated with high RNase activity (such as spleen, pancreas, lungs, and primary B cells) may pose a challenge during isolation of mRNA.…”

“…Analysis of protein expression and dynamics provides insight into how cells react to external stimuli, but correlation with gene expression by measuring changes in mRNA is a central part of the investigation (Henriquez, Zhou, Li, Crawford, & Kaminski, 2017). Effective secretion of cytokines, expression of cell‐surface receptors, and terminal differentiation of cell types are critical for the immune response and clearance of pathogens.…”