Sensitive fluorescent hybridisation protocol development for simultaneous detection of microRNA and cellular marker proteins (in the retina)

Abstract

Nowadays, increasing number of microRNAs are found to have crucial roles in various physiological processes through gene expression regulation via RNA silencing as a result of base pairing with complementary mRNA sequences. To reveal the spatial distribution of microRNA expression in tissues, in situ hybridisation is the only method developed to date. This work aims to provide a novel approach to obtain information on the possible involvement of microRNA-s in regulatory processes under experimental conditions by enhancing fluorescent detection of microRNA labelling. Developing Wistar rats were used as a model system to analyse retinal microRNA expression in the first 3 postnatal weeks. Using cryosections, the crucial elements of optimal labels were (1) the concentration and duration of proteinase K treatment, (2) hybridisation temperature of microRNA probes and (3) temperature of stringency washes. Further improvements made possible to combine our in situ hybridisation protocol with double-label immunofluorescence allowing for the simultaneous detection of microRNA-s with high sensitivity and a neuronal cell marker and/or a synaptic marker protein. Thus, the regulatory microRNA-s can be localised in an identified cell type along with its potential target protein. We believe that our protocol can be easily adapted for a variety of tissues of different origins, developmental stages and experimental conditions.

Analysis of mir-9 Expression Pattern in Rat Retina during Postnatal Development

“…The role of mir-9 in the retinal neurogenesis has been intensively studied in the literature, although little is known about its postnatal distribution and quantity. In our previous in situ methodical study, we saw altered mir-9 expression in the postnatal rat retina [ 51 ]. On the basis of these results, in the present work, we try to explain how mir-9 may contribute to postnatal retinal development by applying more methodical approaches and adding more age groups.…”

Combined microRNA and mRNA detection in mammalian retinas by in situ hybridization chain reaction

“…The application of in situ HCR to miRNA detection should facilitate the identification and characterization of cells expressing specific miRNAs. Combined miRNA and protein detection have been used previously to identify miRNA expressing cells in the retina, using miRNA in situ hybridization with a fluorescent enzymatic detection method and antibody detection of the protein 15 as well as non-neural tissues 14 . The ability to combine miRNA detection with mRNA detection by in situ HCR provides additional options for cell identification and characterization.…”

“…the precipitate can block fluorescence excitation). miRNA in situ methods with fluorescent detection suitable for combined detection have been described, based on enzymatic amplification 10,11,[14][15][16]53 , or a combination of proprietary nucleic acid amplification and enzymatic amplification (RNAscope, which is capable of combined detection of a miRNA with proteins or mRNAs 13,54 ). However, inactivation of enzymes during sequential enzymatic amplification for double label RNA detection may require harsh conditions 55 , and commercial RNAscope reagents are relatively expensive.…”

“…An alternate approach for sequence specific miRNA probes has been to incorporate LNA bases into probes 9 . However, different LNA probes often require different hybridization or wash temperatures to discriminate between related sequences 11,15,56 . The miRNA in situ HCR method employs the same hybridization and wash temperature for multiple probes, and these temperatures are lower than typically used with LNA probes.…”

“…The existence of miRNA families with closely related sequences also complicates the specificity of miRNA detection, but the use of locked nucleic acid (LNA) probes 7 or high stringency wash conditions 6 can discriminate between related miRNAs. Most miRNA in situ hybridization studies have relied on colorimetric enzyme assays for amplification and detection, but detection methods based on fluorescent enzymatic assays 10,11,[13][14][15][16] or radioactive labels 17 have been employed. Fluorescent detection allows combinatorial detection with fluorescent probes for mRNAs 16 or antibodies 15 .…”