Lost in Translation: Detection of mRNA by Flow Cytometry
Flow cytometry is a widely used technology for simultaneous detection of several biomarkers at a single cell level. This quasi-quantitative approach permits the investigator to identify heterogeneity in a sample and make connections between related and unrelated cells. However, the use of flow cytometry has been largely limited to analysis of intra and extracellular proteins and highly abundant DNA sequences. Following transcription, the translation of messenger RNA (mRNA) is regulated through a series of modifications including exportation to the cytoplasm, slicing, capping and incorporation of poly (A) tail. These sequential events subsequently serve as a switch to control protein production in cells (Soh et al., 2018). Additionally, proteins undergo further post-translational modifications that influence their folding, localization, interaction, and functional state (Bürkle 2001). This further increases the complexity of how protein synthesis is regulated (Figure 1). While protein expression and dynamics provide insight into how cells react to external stimuli; understanding the relationship between mRNA and protein expression is also an important part of the investigation. At Flow Contract Site Laboratory (FCSL) we have developed an assay utilizing the PrimeFlowTM (ThermoFisher Scientific) technology to detect cytokine mRNA in frozen human PBMC samples.
Analysis of gene expression has conventionally been performed using reverse transcriptase polymerase chain reaction (RT-PCR), RNA Sequencing (RNA-Seq) and microarray technologies. One of the major limitations of such methods is that the output is a bulk cell average. Since, gene expression may vary among different cell types; investigation of gene expression at a single-cell level provides an attractive alternative (Soh et al 2018).
In 1993, Patterson et al., utilized polymerase chain reaction (PCR) driven amplification and fluorescence in situ hybridization (FISH) to label and detect HIV mRNA in infected cells by flow cytometry (Patterson et al., 1993). This technique opened up the avenue for detection of mRNA at single cell resolution by flow cytometry. Some of the improvements that have been made in recent years include increased sensitivity to detect low abundant mRNA and less repetitive sequences, reduced autofluorescence caused by higher temperature required for hybridization, and finally the expansion of the technology to enable simultaneous detection of intracellular, extracellular protein and RNA. Two commercially available techniques: SmartFlareTM (EMD Millipore) and PrimeFlowTM (ThermoFisher Scientific) have been adapted to flow cytometry.
The SmartFlareTM (EMD Millipore) system utilizes target specific probes which are gold nanoparticles conjugated to oligonucleotides and are taken up by live cells through endocytosis. The probes are designed such that once inside the cell if the probe binds to the mRNA of interest, fluorescence is emitted and can be detected by a flow cytometer. Additionally, after the incubation, the probes exit the cells via exocytosis and the live cells can be used for downstream testing.
The PrimeFlowTM (ThermoFisher Scientific) approach uses fluorescent in situ hybridization that allows specific hybridization of oligonucleotide probes to specific regions across the target mRNA sequence in cells. Then, using the branched DNA technology, the signal is amplified through a series of sequential hybridization steps to form a tree-like structure. Pre-amplifier molecules hybridize to their respective pair of bound oligonucleotide probes (the trunk of the tree), to which hybridizes the amplifier molecules (forming the branches). Finally, multiple label probes hybridize to the amplifiers (the leaves of the tree). Fluorescence can then be detected using a standard flow cytometer (Figure 2).
At FCSL, we have the capabilities to utilize either approach for detection of mRNA by flow cytometry. We recently developed a method for the simultaneous characterization of T lymphocyte subsets and detection of IL-2 and IFN-γ mRNA from frozen human peripheral blood mononuclear cells (PBMC) using the PrimeFlowTM (ThermoFisher Scientific) system. Briefly, cells were stained with cell surface markers followed by fixation and permeabilization of cells to stain with intracellular antibodies. After an additional fixation step, the cells were hybridized with target-specific probes containing 20-40 oligonucleotides (in this case IL-2 and IFN-γ were the targets of interest). Signal amplification was achieved through sequential hybridization steps with preamplifier, amplifier and lastly with fluorochrome-conjugated label probes. After staining, the cells were washed and acquired on a BD FACSCanto™ ten-color cytometer. Our data shows that IL-2 and IFN-γ mRNA can be successfully detected by flow cytometry; while retaining surface staining for T cell characterization (Figure 3).
Additionally, we compared the detection of these cytokine mRNA to conventional approach of intracellular cytokine detection by flow cytometry after fixation and permeabilization of cells. We found that the expression of IL-2 mRNA was significantly higher than IL-2 protein, while, the expression of IFN-γ mRNA and protein was comparable. Additionally, CD4+IFN-γ+IL-2+ and CD8+IL-2+IFN-γ+ populations were observed in RNA assay which were not identified by conventional flow (Figure 4). We hypothesize that the kinetics of IL-2 mRNA and protein expression might be different and therefore a time-course assay would be necessary to elucidate this difference. Since increased mRNA level for cytokines does not always correlate with protein production, it is important to utilize techniques that allow simultaneous measurement of mRNA transcript and protein. This enables investigators to understand T cell responsiveness and identify any aberrant post-transcriptional events. Finally, we have developed this method on frozen PBMC which makes it ideal for clinical samples, when PBMCs from multiple time-points can be isolated, frozen, and later processed for mRNA expression in a single assay, thus reducing variability.
This novel technology is increasingly used among research scientists. Falkenberg et al., have recently developed the method for detecting bovine viral diarrhea virus (BVDV) in infected cell lines using the flow cytometry-based technique (PrimeFlowTM RNA Assay). This study showed that not only it is possible to detect the virus but also quantify the number of infected cells in cell lines with varying viral load. BVDV is an enveloped single stranded RNA virus of the Flaviviridae family, which infects lymphoid tissue. Its characteristics are a transient lymphopenia in the periphery at around day 3, recovery starts on day 9 and complete recovery occurs around day 14 post-infection. Due to this immunomodulation, detection of BVDV viral RNA at a single lymphoid cell level and quantification of number of infected cells is important and had not been possible with PCR based assays (Falkenberg et al., 2017). Early this year the group further published their work on the application of this development to evaluate differential viral distribution within subpopulations of PBMC collected from calves persistently infected with BVDV over time (Falkenberg et al., 2019).
Another investigation of cancer related gene (such as MYCN gene and Wilms’ tumor 1-WT1 gene) expression in neuroblastoma cell lines and acute myeloid leukemia patient samples was performed using the PrimeFlow™ assay and RT-PCR. The group found that both highly overexpressed MYCN gene and moderately expressed WT1 genes were quantifiable and detectable in rare populations (Depreter et al., 2018). Additionally, while the gene expression pattern was comparable to RP PCR, the flow cytometry approach enabled standard membrane staining which remained unperturbed.
In conclusion, PrimeFlow™ RNA assay is a sensitive flow cytometry based technology that allows simultaneous investigation of mRNA expression and protein in heterogeneous samples. The assay is compatible with standard immunostaining, allows association of nucleic acid sequence to immunologic phenotype, enumerates gene expression heterogeneity at a single-cell level compared to conventional approach which is limited to bulk population average and eliminate the constraints of lack of antibodies for certain non-coding mRNA targets. The combination of flow cytometry to molecular based approach decreases analytical time which had previously required the two-step process of sorting homogenous population using cell sorters which were then subjected to genomic assays.
FCSL is a contract flow lab that provides high throughput and high capacity flow cytometry services, running multiple flow cytometers with up to 10 color antibody panels daily. We are proficient in processing a multitude of specimen types including whole blood, frozen PBMCs along with cell culture and tissue processing capabilities. Our flexibility in handling so many specimen types allow for the support of a wide range of flow cytometry assays including: immunophenotyping/lymphocyte subset analysis, receptor occupancy, functional assays and cell viability/apoptosis measurements. Our expert staff is always available to help guide you through these tests and we welcome clients to visit our facility. We encourage sponsor engagement throughout the process. Contact us for more information!
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