In response to external stimuli, differential regulation of RNA transcription leads to changes in protein level. However, the levels of RNA and protein products of specific genes may vary at any given time point. Using current methods such as qPCR or microarrays, researchers must choose between measuring mRNA or protein and cannot measure both simultaneously due to the limitations of prevailing techniques. PrimeFlow® RNA can reveal the unique kinetics of mRNA and protein expression in different cell subsets, and elucidate an unbiased correlation as it changes over time in response to the stimulus.
Correlation and kinetics of IFNγ and TNFα transcription and translation in lymphocyte subsets
Intracellular staining and flow cytometric analysis of lymphocytes is commonly used to assess cytokine production at the single-cell level in heterogeneous samples. Here, PrimeFlow® RNA is used in combination with intracellular antibody staining to study the kinetics of the transcription and translation of IFNγ and TNFα in lymphocytes.
IFNγ mRNA was upregulated in CD8+ and CD8- lymphocytes within 1 hour after stimulation, while protein levels were not detected until 2 hours. Both IFNγ mRNA and protein were maintained for the next 3-4 hours (top panel). In contrast, TNFα mRNA and protein were both upregulated within 1 hour after stimulation and expression was maintained in CD8+ cells while expression in CD8- cells peaked between 1-2 hours and then decreased over the next 4 hours, with the decrease in mRNA preceding the decrease in protein (bottom panel).
Using the PrimeFlow® RNA, we find that induction of IFNγ and TNFα mRNA and protein exhibit unique kinetics and that TNFα protein and mRNA are differentially regulated in CD8+ and CD8- lymphocytes. This new PrimeFlow® RNA Assay enables the study of gene expression at the single-cell level in heterogeneous samples without the need for sorting specific subsets and the ability to compare and contrast the kinetics of mRNA and protein induction.
Kinetics of IFNy and TNFα transcription and translation measured by PrimeFlow® RNA Assay
Normal human peripheral blood mononuclear cells were stimulated with PMA and Ionomycin for 0-5 hours. Using PrimeFlow&trade: RNA Assay, cells were fixed, permeabilized, and intracellularly stained with antibodies for CD8, IFNγ, and TNFα. Next, cells underwent a series of hybridization steps to label mRNA for IFNγ and TNFα. Viable CD8+ and CD8- cells in the lymphocyte gate were used for analysis. Figures 1A and 1C reveal the kinetics of IFNγ and Figures 1B and 1D illuminate the kinetics of TNFα.