MicroRNA in situ hybridization for formalin fixed kidney tissues. J Vis Exp 2013 Nov 30(81)
Date
12/12/2013Pubmed ID
24326287Pubmed Central ID
PMC3992125DOI
10.3791/50785Scopus ID
2-s2.0-84888876317 (requires institutional sign-in at Scopus site) 13 CitationsAbstract
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 MMESH terms used to index this publication - Major topics in bold
AnimalsColorimetry
Formaldehyde
In Situ Hybridization
Kidney
MicroRNAs
Microtomy
Rats
Tissue Fixation
