ViewRNA Data Specifications
Assay Performance Highlights
ViewRNATM ISH Cell Assay
|Sample Type||Adherent and suspension cells*|
|Limit of Detection (LOD)||1 RNA molecule/cell|
|Plex Level||1 to 4-plex|
|Assay Format||Slides, 4-well chamber, 24-well plates (cover slips)|
|Automation-compatible||Fluorescence microscope or imaging system|
The ViewRNA ISH Cell Assay for multiplex fluorescence RNA in situ hybridization, 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.
Validate and Localize RNA Targets Identified by Sequencing
RNA targets identified by sequencing were validated using ViewRNA ISH Cell Assay in cultured neurons. Cajigas I. J., et al. The local transcriptome in the synaptic neuropil revealed by deep sequencing and high-resolution imaging. Neuron 74(3):453-66 (2012).
Figure 1. Cultured hippocampal neurons: mRNA transcripts (Camk2a and Shank1 - red) visualized using ViewRNA ISH Cell Assay. Map2 protein (to generate a mask that outlines the dendrites) - immunostained.
Scale bar = 20 µm.
Visualization of Individual RNA Molecule at Single Cell Level in Differentiating Cells
We profiled the expression pattern of a myogenin marker gene in differentiating C2C12 myoblastic cells. The results demonstrated the profound changes in gene expression profiles in the course of myotube formation and also highlighted the differences between undifferentiated and committed cells in the mixed population.
Figure 2. Using fluorescence RNA in situ hybridization assay to study myogenin expression in differentiating cells. C2C12 cells cultured in 96 well plate were formaldehyde fixed at 0, 2 and 5 days differentiation. The fixed cells were permeabilized and probed for myogenin expression. Bright field images are in the top row and fluorescent images in the bottom row with myogenin mRNA (green) and DAPI nuclear stain (blue). Bright field image is in different field than fluorescent images.
Infectious Diseases—Analysis of Viral Genes in Cells
The ViewRNA ISH Cell Assay is well suited to investigate the presence, location, and trafficking of viral genes within individual cells and the host response to virus and other infectious disease agents (Figure 2, 3).
Figure 3. Detection of H1N1 Influenza A RNA migration in MEF cells using RNA fluorescence in situ hybridization. Murine embryonic fibroblasts (MEFs) were incubated on ice with influenza A virus (H1N1, PR8 strain). At time 0, virus was removed and 37Â° C media was added. At the indicated time points after warming, the cells were fixed and processed with the ViewRNA ISH Cell Assay kit and a probe set against the nucleoprotein (NP) viral genomic segment (green). DNA is stained blue with DAPI. Time-dependent nuclear translocation of the NP genome is seen at 90 minutes post infection, and by 180 minutes the nuclear export of newly synthesized viral genomes to the cytosol is also observed. Data courtesy of Dr. Abraham L. Brass, MD, PhD, Ragon Institute of MGH, MIT and Harvard
Figure 4. Multiplex detection in Huh-7 cells using the RNA FISH assay. Multiplex fluorescence RNA in situ detection of HCV viral genomic RNA (green) and 18S RNA (red) in Huh-7 cells lacking (-HCV) or containing (+HCV) an HCV replicon (Ikeda et al., 2002, J. Virol., 76: 2997-3006). In both panels, nuclei are stained with DAPI (blue).
Quantitation of siRNA Knockdown
Perform siRNA knockdown studies with high specific signal and low background. Direct labeling of the knockdown target RNA allows fast and easy monitoring of the siRNA knockdown efficiency and study of off-target effects. Here we show the siRNA knockdown of HPRT1 (green) in HeLa cells. ACTB (red) was used as a control gene and no knockdown was observed.
Figure 5. HPRT1 knockdown in HeLa cells using the RNA FISH assay. HeLa cells were transfected with various concentrations of HPRT1 siRNA. Following transfection, cells were fixed and analyzed for HPRT1 (green) and ACTB (red). The images clearly show partial knockdown of HPRT1 at the 0.04 nM siRNA concentration and complete knockdown at the 10 nM siRNA concentration and no effect on the ACTB which was used as a control gene.
In Situ RNA Quantitation in Time Course Studies
Visualize and quantitate expression of multiple transcripts following induction using RNA FISH assay. HeLa cells were treated with PMA and the induction of IL-6 (red), IL-8 (yellow) and ACTB (control gene, green) were followed over time. Heterogeneity of the interleukin responses is clearly evident, while ACTB expression is consistent across all treatment conditions. IL-6 shows peak signal from 0.5 to 2 hours, while IL-8 signal gradually increases with peak signals at 8 hours.
Figure 6. Time course induction of interleukins in HeLa cells using RNA FISH assay. HeLa cells were treated with 100ng/mL of PMA for 0, 0.5, 1, 2, 4, and 8 hours. Cells were fixed, permeabilized and probed for IL-6, IL-8 and ACTB mRNA. Expression level of IL-6 (red), IL-8 (yellow) and ACTB (green) were detected in PMA-treated HeLa cells at each time points. Nuclei (blue) are stained with DAPI.
Robust Signal in Suspension Cells
ViewRNA ISH Cell Assay provides robust results in adherent as well as suspension cells. Jurkat cells, T lymphocyte cells were probed for HPRT1 (green), PPIB (yellow) and GAPDH (red), representing low to high expression levels respectively. Excellent signal to noise was observed.
Figure 7. Robust signal in suspension cells using RNA in situ hybridization assay. Jurkat cells were fixed, permeabilized and assayed with probes sets for HPRT1 (green), PPIB (yellow) and GAPDH (red). Nuclei (blue) are stained with DAPI. The expression of these genes can be visualized under fluorescence microscope.
ViewRNA Complete Bibliography Click here
- Cajigas I. J., et al. The local transcriptome in the synaptic neuropil revealed by deep sequencing and high-resolution imaging. Neuron 74(3):453-66 (2012).
- Feeley E. M., et al. IFITM3 inhibits influenza A virus infection by preventing cytosolic entry. PLoS ONE 7(10):E1002337 (2011).
- Garaigorta U., et al. Hepatitis C virus (HCV) induces formation of stress granules whose proteins regulate HCV RNA replication and virus assembly and egress. J Virol 86(20):11043-56 (2012).
- Lee K., et al. Precursor miR-886, a novel noncoding RNA repressed in cancer, associates with PKR and modulates its activity. Rna 17(6):1076-89 (2011).
- Louzada S., Adega F., Chaves R. Defining the sister rat mammary tumor cell lines HH-16 cl.2/1 and HH-16.cl.4 as an in vitro cell model for Erbb2. PLoS ONE 7(1):e29923 (2012).
- Taylor A. M., et al. Axonal mRNA in uninjured and regenerating cortical mammalian axons. J Neurosci 29(15):4697-707 (2009).
- Yu M., et al. RNA sequencing of pancreatic circulating tumour cells implicates WNT signalling in metastasis. Nature (2012).