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MicroRNA in situ hybridization for formalin fixed kidney tissues. J Vis Exp 2013 Nov 30(81)

Date

12/12/2013

Pubmed ID

24326287

Pubmed Central ID

PMC3992125

DOI

10.3791/50785

Scopus ID

2-s2.0-84888876317   7 Citations

Abstract

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes(1), has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

Author List

Kriegel AJ, Liang M

Authors

Alison J. Kriegel PhD Associate Professor in the Physiology department at Medical College of Wisconsin
Mingyu Liang PhD Center Director, Professor in the Physiology department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Animals
Colorimetry
Formaldehyde
In Situ Hybridization
Kidney
MicroRNAs
Microtomy
Rats
Tissue Fixation
jenkins-FCD Prod-484 8aa07fc50b7f6d102f3dda2f4c7056ff84294d1d