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ViewRNA miRNA ISH Cell Assay- Data/Specifications

Assay Performance Highlights

ViewRNATM miRNA ISH Cell Assay

Sample Type Adherent and suspension cells
Species Any
Sensitivity Single-molecule detection
Plex Level 1 miRNA and up to 2 mRNAs
Assay Format 24-well plates*
96-well optical bottom imaging plate
Detection Fluorescent
Instrumentation Fluorescence microscope or imaging system

* 24-well plates - cover slips placed on the bottom of the 24-well plate


Simultaneous detection of miRNA, mRNA and/or protein in dissociated mouse hippocampal cultures

MAP2 and GFAP markers

Dissociated mouse hippocampal culture was stained by immunofluorescence (IF) for Map2 (neuronal cells) and Gfap (glia cells) markers to positively identify neurons and glial cells in the mixed culture (Figure 1). The same mixed culture was subjected to miRNA in situ hybridization using the ViewRNA miRNA ISH Cell Assay and probed against a neuronal specific miRNA target.

 

miRNA_mRNA_in_situ_hybridization_FISH

*Data courtesy of UCLA

Figure 1. miRNA in situ detection of neuron-specific targets in dissociated mouse hippocampal cultures. Visualization of neuronal and glia cells by RNA in situ hybridization and IF. Neuronal specific miRNA was stained using the ViewRNA miRNA ISH Cell Assay. Map2 and Gfap were stained using IF. miRNA target (red—neuronal marker), Map2 protein (light blue—neuronal marker) and Gfap protein (green—glia marker).


Specificity of the mRNA probe in the mRNA in situ hybridization assay

visualize_miRNA_mRNA

Accurately and precisely study and visualize miRNA and mRNA of various expression levels. The assay has single-molecule RNA sensitivity – where one punctated dot corresponds to one molecule of miRNA or mRNA target.

 

visualize miRNA and mRNA

Figure 2: Multiplex miRNA and mRNA in situ hybridization assay in adherent cells. Simultaneous detection of miRNA (Fast Red Substrate – Cy3/550 channel) and mRNA (in FITC channel and/or in Cy7 channel) expression in any given cell. Shown are duplex assays showing expression of different miRNA and mRNA targets in HeLa cells.


Monitor siRNA knockdown using the miRNA in situ hybridization assay

siRNA knockdown using miRNA in situ hybridization

Simplify gene expression studies by visualizing siRNA transfection and gene expression in the same sample. The miRNA in situ hybridization assay allows the monitoring of siRNA and the mRNA of interest for fast and easy analysis of siRNA knockdown efficiency.

 

siRNA knockdown using miRNA in situ hybridization

Figure 3. Visualize and quantitate siRNA knockdown efficiency in situ. HeLa cells were transfected with various concentrations of GAPDH siRNA. Following transfection, cells were analyzed for GAPDH siRNA (red) and GAPDH mRNA (green). The images clearly show that the level of GAPDH knockdown is directly proportional to the level of siRNA present in the cells as detected by the View miRNA ISH Cell Assay. Nuclei (blue) were stained with DAPI.


Visualization and quantitation of the miRNA/mRNA in situ hybridization assay

visualization and quantitation of the miRNA in situ hybridization assay

The miRNA in situ assay results can be analyzed using two analysis software: MetaMorph and CyteSeer. In this example the expression levels of four miRNA targets of various expression levels, were analyzed in HeLa cells. The results were imaged using Olympus fluorescence microscope, visualized using MetaMorh (Figure 4A) and quantified using CyteSeer (Figure 4B).

 

visualization and quantitation of the miRNA in situ hybridization assay

Figure 4. Analysis of View miRNA ISH Cell Assay Data with CyteSeer Software. Expression of several microRNA targets in HeLa cells were assessed using the ViewRNA miRNA ISH Cell Assay. The images were captured with similar exposure time using an Olympus fluorescent microscope equipped with a cooled-CCD camera, 20X/0.75 N.A. objective lens, and filter sets for DAPI and Cy3 channels.

A. MetaMorph Imaging Software (Molecular Devices) was used to visualize the acquired images, subtract non-specific background and overlay signals from different channels to generated merged images that display the different expression levels of the 4 targets in HeLa cells.

B. CyteSeer (ValaSciences) was employed to quantify the mean total integrated Fast Red signal intensity per cell for each of these 4 targets. As the bar graph indicated, good correlation was obtained between the visual and quantitative data.


miRNA/mRNA in situ hybridization assay concordance with QuantiGene 2.0 miRNA Assay

Assay Concordance

The ViewRNA miRNA ISH Cell Assay can be used to validate QuantiGene 2.0 miRNA Assay results. To evaluate concordance between these two assays, the expression levels of 10 different miRNA targets in HeLa cell was quantified using the QuantiGene 2.0 miRNA Assay. Separately, in situ miRNA analysis of the same 10 miRNA targets was performed using the ViewRNA miRNA Assay and the mean of total signal intensity for each miRNA target was quantified using CyteSeer software.

 

Assay Concordance

Figure 5. ViewRNA miRNA Assay and QuantiGene 2.0 miRNA Assay data concordance. A regression analysis based on MAQC method, in which the miRNA copy #/log2 was plotted as a function of the mean of total miRNA signal intensity/log2 gave a correlation coefficient (R2 value) of 0.7933, indicating good concordance between the two data sets obtained by two independent methods.