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A Method to Pre-Screen Rat Mammary Gland Whole-Mounts Prior To RNAscope

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Journal of Mammary Gland Biology and Neoplasia Aims and scope Submit manuscript

Abstract

RNAscope is a quantitative in situ gene expression measurement technique that preserves the spatial aspect of intact tissue; thus, allowing for comparison of specific cell populations and morphologies. Reliable and accurate measurement of gene expression in tissue is dependent on preserving RNA integrity and the quantitative nature of RNAscope. The purpose of this study was to determine if the quantitative nature of RNAscope was retained following processing and carmine staining of mammary gland whole-mounts, which are commonly used to identify lesions, such as hyperplasia and ductal carcinoma in situ (DCIS). We were concerned that handling and procedures required to visualize microscopic disease lesions might compromise RNA integrity and the robustness of RNAscope. No effect on the quantitative abilities of RNAscope was detected when mammary gland whole-mounts were pre-screened for lesions of interest prior to RNAscope. This was determined in comparison to tissue that had been formalin-fixed and paraffin embedded (FFPE) immediately after collection. The ability to pre-screen whole-mounts allowed unpalpable diseased lesions to be identified without labor-intensive serial sectioning of tissue samples to find diseased tissue. This method is applicable to evaluate mammary gland whole-mounts during normal mammary gland development, function, and disease progression.

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Data Availability

Data used and analyzed for this study are not publicly available because they are saved on private servers; however, these data are available from the corresponding author upon request.

Abbreviations

FFPE:

Formalin-fixed and paraffin embedded

RNA ISH:

RNA in situ hybridization

DMBA:

7,12-Dimethylbenz(a)anthracene

WF:

Wistar Furth

DCIS:

Ductal carcinoma in situ

IDC:

Invasive ductal carcinoma

H&E:

Hematoxylin and Eosin

DapB:

Four-hydroxy-tetrahydrodipicolinate reductase

Mier3:

Mesoderm induction early response family member 3

Polr2A:

RNA Polymerase II Subunit A

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Funding

The University of Louisville Integrated Programs in Biomedical Sciences and the Department of Biochemistry and Molecular Genetics.

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Authors and Affiliations

  1. Department of Biochemistry & Molecular Genetics, University of Louisville School of Medicine, Louisville, KY, USA

    Emily L. Duderstadt & David J. Samuelson

  2. Department of Pathology, University of Louisville School of Medicine, Louisville, KY, USA

    Mary Ann Sanders

  3. James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY, USA

    David J. Samuelson

Authors
  1. Emily L. Duderstadt
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  2. Mary Ann Sanders
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  3. David J. Samuelson
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Contributions

ELD administered DMBA, performed tissue dissection and whole-mount preparation, sectioned and stained tissue, conducted RNAscope assays and quantitative measurement of images, analyzed data, prepared figures, and wrote this manuscript. MAS provided pathology expertise and wrote this manuscript. DJS designed experiments, analyzed data, prepared figures, and wrote this manuscript.

Corresponding author

Correspondence to David J. Samuelson.

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Ethics Approval

All animal protocols were approved by the University of Louisville Animal Care and Use Committee (current protocol #20,715).

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The authors declare no competing interests.

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Duderstadt, E.L., Sanders, M.A. & Samuelson, D.J. A Method to Pre-Screen Rat Mammary Gland Whole-Mounts Prior To RNAscope. J Mammary Gland Biol Neoplasia 26, 113–120 (2021). https://doi.org/10.1007/s10911-021-09484-5

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  • DOI: https://doi.org/10.1007/s10911-021-09484-5

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