Multiplex In Situ Hybridization in the Study of Acute Kidney Injury : Multiplex In Situ Hybridization in AKI.

Abstract

Recently developed in situ hybridization (ISH) methods, such as RNAscope™, have greatly expanded the accessibility and usefulness of ISH in biomedical research. Among many other advantages over traditional ISH, these newer methods enable the simultaneous use of multiple probes, including combination with antibody or lectin staining. We herein illustrate the application of RNAscope™ multiplex ISH in the study of the adapter protein Dok-4 in acute kidney injury (AKI). Specifically, we used multiplex ISH to define the expression of Dok-4 and some of its putative binding partners, together with nephron segment markers, as well as markers of proliferation and tubular injury. We also illustrate the use of QuPath image analysis software to perform quantitative analyses of multiplex ISH. Furthermore, we describe how these analyses can exploit the uncoupling of mRNA and protein expression in a knockout (KO) mouse created by CRISPR/CAS9-mediated frame shift to carry out highly focused molecular phenotyping studies at the single-cell level.

• Authors: Agnieszka Masztalerz, Alex Gregorieff, Serge Lemay, Tomoko Takano
• Journal: Methods in molecular biology (Clifton, N.J.) (Methods Mol Biol) Vol. 2664 Pg. 217-232 ( 2023) ISSN: 1940-6029 [Electronic] United States
• PMID: 37423993 (Publication Type: Journal Article)
• Copyright: © 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Chemical References

• RNA, Messenger

Topics

• Mice
• Animals
• In Situ Hybridization
• Acute Kidney Injury (genetics, metabolism)
• Nephrons (metabolism)
• RNA, Messenger (genetics)
• Staining and Labeling
• Kidney (metabolism)
• Reperfusion Injury (metabolism)