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A Method to Pre-Screen Rat Mammary Gland Whole Tissue Mounts followed by RNAscope in situ hybridization
David J. Samuelson
University of Louisville
Corresponding Author
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Background: RNA in situ hybridization is an extremely useful gene expression analysis technique that preserves the spatiotemporal nature of tissue and allows for evaluation of specific cell populations and morphologies. This technique is especially useful in evaluating expression changes in disease progression models. Tissue processing procedures used to identify pathological disease morphologies in situ could compromise RNA integrity and the reproducibility and quantitative accuracy of RNA in situ hybridization assays.
Methods: A combinatorial approach to pre-screen rat mammary gland tissue whole mounts for hyperplastic and malignant lesions that were not visually discernible without staining was used. This pre-screening process was followed by an RNA in situ hybridization analysis method known as RNAscope.
Results: We show that there are no differences in the quantitative nature of RNAscope assays between tissue that was immediately formalin-fixed and paraffin embedded (FFPE), which is recommended by the manufacturer, and tissue that was whole mounted and pre-screened for lesions of interest prior to RNAscope.
Conclusions: Preserving the integrity of RNA and quantitative nature of the RNAscope assay is important, as it allows unpalpable lesions to be directly identified, bypassing a need for labor-intensive serial sectioning of FFPE tissues to find lesions. This method is applicable to any epithelial-based disease progression model using whole-tissue mounts.
Keywords
RNAscope, tissue screening, quantitative biology
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This is a preprint. It has not completed peer review.
Research
A Method to Pre-Screen Rat Mammary Gland Whole Tissue Mounts followed by RNAscope in situ hybridization
David J. Samuelson
University of Louisville
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 07 Aug, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Animal Science
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: RNA in situ hybridization is an extremely useful gene expression analysis technique that preserves the spatiotemporal nature of tissue and allows for evaluation of specific cell populations and morphologies. This technique is especially useful in evaluating expression changes in disease progression models. Tissue processing procedures used to identify pathological disease morphologies in situ could compromise RNA integrity and the reproducibility and quantitative accuracy of RNA in situ hybridization assays.
Methods: A combinatorial approach to pre-screen rat mammary gland tissue whole mounts for hyperplastic and malignant lesions that were not visually discernible without staining was used. This pre-screening process was followed by an RNA in situ hybridization analysis method known as RNAscope.
Results: We show that there are no differences in the quantitative nature of RNAscope assays between tissue that was immediately formalin-fixed and paraffin embedded (FFPE), which is recommended by the manufacturer, and tissue that was whole mounted and pre-screened for lesions of interest prior to RNAscope.
Conclusions: Preserving the integrity of RNA and quantitative nature of the RNAscope assay is important, as it allows unpalpable lesions to be directly identified, bypassing a need for labor-intensive serial sectioning of FFPE tissues to find lesions. This method is applicable to any epithelial-based disease progression model using whole-tissue mounts.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5