Abstract
miRNA are short non-coding RNA which inhibit translation of mRNA. miRNA regulate several cellular processes. Certain miRNA are known to induce oncogenesis. miRNA can be measured by real-time PCR and be imaged using a combination of in situ hybridization (ISH) and quantum dots (QD). The advantage of using quantum dots is that several miRNA can be simultaneously measured using multiplexed QD. Additionally, miRNA can be visualized in different regions of the tissue. Since miRNA are biomarkers of various disease states, miRNA can be visualized and quantitated in tissue sections for diagnostic and prognostic purposes. Here we describe ISH-QD analysis of tissue sections. Tissue sections from xenografts or clinical specimens are used. These are deparaffinized, treated with Proteinase K and hybridized with a biotin-probe to specific to the miRNA. The in situ hybridization is performed by labeling the biotin-probes and followed by labeling with streptavidin tagged quantum dots. Image acquisition of the quantum dots is performed and analyzed for the miRNA expression levels. Combining ISH and QD gives a powerful tool to detect miRNA in different cells of the tissue.
Keywords: miRNA, in situ hybridization, Multiplexed quantum dots, Cancer, Cancer associated stroma, Cellular compartments, Biomarkers, Tissue staining
Background
miRNAs can be easily detected by quantitative real-time PCR or Northern blotting. However, imaging miRNAs has been challenging. Recent advances in quantum dots imaging have made it possible to determine expression of miRNAs in tissues. Using this process, miRNAs can be visualized in different compartments of a tissue, such as tumor, stroma, immune cells, etc. Additionally, miRNAs can be multiplexed to determine co-localization of miRNAs which mediate specific processes, in different cellular regions. Different tissues can be used for ISH-QD such as tissues from xenografts or human clinical samples. Tissues from animal studies are formalin fixed and paraffin embedded. These tissues were used for ISH-QD analysis (Figure 1). Figure 1. In situ hybridization coupled to Quantum dots labelling for visualization of miRNA. In the ISH-QD protocol, miRNA are detected on formalin fixed paraffin embedded tissue sections using biotinylated miRNA probes binding to streptavidin-conjugated QDs. The specific QD gives a specific fluorescent signal which is quantified using the Inform v1.3 software.
Materials and Reagents
Equipment
Software
Procedure
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Data analysis
Once data acquisition is performed the images are deconvoluted or unmixed to extract the specific signal by a specified QD. A spectral library was built for 565 and 625 nm. The spectral library was used to unmix the cube. Autofluorescence was reduced using the Real Component Analysis plug-in software. The images after unmixing represented the distribution of QDs on the tissue. Signal was quantified using Inform v1.3 software. The software discriminates cancer and stromal (non-cancer) areas. The software also discriminates between nuclear and cytoplasmic area based on the DAPI stain. Using this software the QD signal from the tumor and stromal sections can be determined and quantified. Numerical values are generated for the image and the cytoplasmic component of the tumor and stromal regions is used for plotting the data. The data is graphically plotted using Graphpad Prism software. Data distribution was depicted in box plot formats. Statistical analysis of tissue array was performed using a non-parametric Wilcoxon rank sum test. Values of P < 0.05 were considered to be statistically significant.
Notes
Recipes
Acknowledgments
This procedure has been published in part in the paper by Hu et al., 2011 and Josson et al., 2015. The in situ hybridization procedure has been optimized from the Exiqon ISH protocol. Grant support for this work is from P01-CA98912, DAMD-17-03-02-0033, RO1-CA122602 (L.W.K. Chung).
References
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