Detection and Differentiation of Multiple Viral RNAs Using Branched DNA FISH Coupled to Confocal Microscopy and Flow Cytometry

Figures

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  Figure 1.. Detection of target RNAs using branched DNA technology. Branched DNA technology for RNA detection can be coupled with confocal microscopy or flow cytometry. Target RNAs are first bound by pairs of Target Probes. Typically, twenty sets of target probe pairs are designed per target RNA. The Pre-Amplifier DNA only binds target probe pairs that are bound to target RNAs in the correct orientation; this greatly limits the signal to noise ratio. Pre-Amplifier DNAs are then bound by Amplifier DNAs and subsequently by Label Probes. This process results in the labeling of target RNAs by up to 8,000 fluorophores.

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  Figure 2.. 40 ± 1 °C incubator setup. Two heat blocks are stored in the incubator to regulate the temperature of RNA FISH flow cytometry samples. A thermometer is kept inside to confirm the digital temperature readings.

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  Figure 3.. Reagents setup for confocal microscopy. Huh7.5.1 cells are plated onto Micro Cover Glass within a 12-well tissue culture plate. These cells are infected following a 24 h incubation to allow cell adherence to the glass. Following infection, the cells are fixed, stained for protein and RNA using the ViewRNA Cell Plus Assay kit, all within the 12-well plate. The Micro Cover Glass is then carefully transferred to a Microslide spotted with PermaFluor/DAPI using fine forceps.

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  Figure 4.. Identification of co-infected cells by PrimeFlow RNA FISH. Huh7.5.1 cells were infected with JFH1-CA, JFH1-WT or co-infected at multiplicities of infection equal to one virus per cell with each virus. Infected cells were incubated for 72 h before labeling viral RNAs using PrimeFlow. Analysis and compensation was performed using FlowJo.

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  Figure 5.. Analysis of viral RNA-protein colocalization using ViewRNA Cell Plus. Huh7.5.1 cells were co-infected with JFH1-WT and JFH1-CA on Micro Cover Glass for 72 h. Cells were stained for HCV core protein and both viral RNAs using the ViewRNA Cell Plus Assay. Quantification of colocalization was performed using Volocity software. Scale bars are 2.5 μm in length.