In situ miRNA Expression Profiling Using Multispectral Imaging
Since the discovery of the RNA interference phenomenon, there has been significant interest in studying functional analysis, expression profiling and up- and down- regulation of miRNAs in an effort to better understand specific biological function(s). The ultimate goal of such studies is to develop more effective therapeutics and improved diagnostics for the multitude of diseases that have been linked to abnormal miRNA expression. Until very recently, such studies have been limited to the estimation of expression levels of miRNAs using PCR (polymerase chain reaction) or microarrays.
Contextual detail necessary to elucidate mechanisms of up- or down-regulation and/or distribution of miRNAs in a particular tissue is not feasible using these techniques, making it necessary to employ in situ methods. In situ studies offer the benefit of visualizing miRNAs associated with specific zonal regulations, given that organs have heterogeneous cellular constituents that give rise to differential expression of miRNAs. Such differential expression levels within a tissue add to the complexity of the studies, often necessitating the use of multiple markers to accurately identify specific information on the interplay of zonal expression levels of miRNAs.
Conventional immunohistochemistry techniques are limited by the degree to which multiple miRNAs can be studied on any given sample given the spatial and spectral overlap associated with imaging multiple miRNAs in a given sample. Such limitations are even more pronounced in fluorescence assays, as even single labels are typically masked by the confounding effects of autofluorescence. These hurdles are overcome by the use of CRi’s Nuance™ multispectral imaging system, which effectively identifies and quantitates multiple miRNA signatures in a given sample using either brigthfield or fluorescence immunohistochemistry.
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Figure 1. miRNA detection in a control vs. labeled human hippocampus specimen. Top row includes a control image acquired using a 20x magnification (a), an image of unmixed FFPE autofluorescence (b), an unmixed image of lipofuscin granules (c), an unmixed image of Cy3 negative control (d) and a spectrally unmixed composite image with the autofluorescence removed. Bottom row includes a conventional RGB image of the Cy3-miRNA 320 acquired using a 20x magnification (a), an image an image of unmixed FFPE autofluorescence (b), an unmixed image of lipofuscin granules (c), an unmixed image of Cy3 expression (d) and a spectrally unmixed composite image of lipofuscin (purple) and miR320 (green) with the autofluorescence removed.
Multispectral Imaging of miRNAs in Parkinson’s and Alzheimer’s Disease
Great strides have been taken in the study of miRNA expression profiling in an effort to elucidate pathways involved in neurodegenerative diseases. We provide a specific example in demonstrating how multispectral imaging can improve the visualization and quantitation of miRNA expression in the hippocampus of diseased tissue (Figure 1).
Multispectral imaging (MSI) is an approach that optimizes the opportunities for multiplexing while at the same time overcoming the effects of autofluorescence on detectability and reliable quantitation. The CRi Nuance multispectral imaging system combines a powerful liquid-crystal tunable filter, a high-end CCD camera and unmixing algorithms to enable fast and accurate unmixing of signals that may overlap both spatially and spectrally, separating them faithfully, without cross-talk.
Published in BioTechniques Application Forum Vol 45, No 5, 2008
