ViewRNA: How it Works
Fluorescence RNA In Situ Hybridization—Assay Work Flow
The ViewRNA ISH Cell Assay is a direct fluorescence RNA in situ hybridization method that uses proprietary chemistry for the target specific probe sets (RNA FISH probes) and branch DNA signal amplification (bDNA) for detection of specific signal. Multiplexing of RNA targets is possible by using independent but compatible signal amplification systems. The fluorescence RNA in situ hybridization assay has four main steps: sample preparation, target hybridization, signal amplification, and detection.
Sample Preparation: Suspension or adherent cells are fixed with formaldehyde and permeabilized using protease to allow target accessibility.
Target Hybridization: A target-specific probe set hybridizes to the target RNA. Subsequent signal amplification is predicated on specific hybridization of adjacent Probe Set oligonucleotide pairs. For clarity, only a single oligonucleotide pair is shown per target mRNA. However, a typical Probe Set contains 20 oligonucleotide pairs.
Signal Amplification: Signal amplification, using bDNA technology, is achieved via a series of sequential hybridization steps. The PreAmplifier molecules hybridize to their respective pair of bound Probe Set oligonucleotides, then multiple Amplifier molecules hybridize to their respective PreAmplifier. Next, multiple Label Probe oligonucleotides conjugated to the fluorescent dye hybridize to the corresponding Amplifier molecule. A fully assembled signal amplification “tree” has 400 binding sites for each Label Probe. When all target-specific oligos in the Probe Set bind to the target mRNA transcript, an 8,000 fold amplification occurs for that one transcript.
Fluorescent Detection: Target mRNAs are visualized using a standard fluorescence microscope with the corresponding filter sets (EX: 488, 550 and 650 nm). If using the ViewRNA ISH Cell 740 Module, a 740 filter is required.
Choosing Target Specific Probe Sets for the Fluorescence RNA in situ Hybridization Assay (RNA FISH)
Compatible Sample Types for the Fluorescence RNA In Situ Hybridization Assay (RNA FISH)
The ViewRNA ISH Cell Assay has been optimized for analyzing adherent and suspension cultured cells. However other samples may be used - like circulating tumor cells, cells isolated from blood or other body fluids. We do not provide a protocol for the analysis of target RNAs from those starting materials. However we do recommend that you follow the protocol for analyzing suspension cells and you optimize as necessary.
Specificity of the Target Specific Probe Sets (RNA FISH Probes)
To demonstrate the target specific probe sets specificity we utilized 18S rRNA as a model. The target specific probe set is comprised of 20 oligonucleotide pairs. In order for a signal amplification branch to bind to the target RNA, both oligos from the oligonucleotide pair need to hybridize side by side to the target RNA (a). To illustrate the specificity of the gene specific probe sets, we used only one of the oligonucleotide pairs within the 18S rRNA Probe Set (b, c). Signal levels were near background when either one of the oligonucleotide pairs was eliminated from the target specific probe set (b, c). Thus, if a target oligonucleotide binds to an unintended sequence nonspecifically, it will not yield a significant signal.
Figure 1: High specificity of the target RNA probe sets (RNA FISH probes). HeLa cells were fixed, permeabilized and probed for 18S RNA (green), using a complete Probe Set (a) or partial Probe Sets consisting of only the right or left size of the Probe Set oligonucleotide pairs (b, c). Nuclei (blue) are stained with DAPI.
Single Transcript Sensitivity of the Fluorescence RNA in situ Hybridization Assay (RNA FISH)
To demonstrate single transcript sensitivity of the multiplex fluorescence RNA in situ hybridization assay, one dot is equivalent to one target molecule, two Probe Sets were designed to target different regions of the ERBB2 (Her-2) mRNA. One Probe Set targeted the region from exons 2-9 (l550-fluorophore) and the other Probe Set targeted the region from exons 10-20 (488-fluorophore). The image was captured with a slight off-set to enable visualization of both signals. If one dot is equivalent to detection of one target, we would expect to see pairs of red and green dots as is evident in the image.
The image was captured with a slight off-set to enable visualization of both signals. If one dot is equivalent to detection of one target, we would expect to see pairs of red and green dots as is evident in the image.
Figure 2. Single transcript sensitivity of the multiplex fluorescence RNA in situ hybridization assay. Each set of red and green “dots” side by side corresponds to a single copy of Her-2 mRNA. Nuclei (blue) are stained with DAPI.