Analysis of microRNA expression by in situ hybridization with RNA oligonucleotide probes

Abstract

In situ hybridization is an important tool for analyzing gene expression and developing hypotheses about gene functions. The discovery of hundreds of microRNA (miRNA) genes in animals has provided new challenges for analyzing gene expression and functions. The small size of the mature miRNAs (∼20-24 nucleotides in length) presents difficulties for conventional in situ hybridization methods. However, we have developed a modified in situ hybridization method for detection of mammalian miRNAs in tissue sections, based upon the use of RNA oligonucleotide probes in combination with highly specific wash conditions. Here we present detailed procedures for detection of miRNAs in tissue sections or cultured cells. The methods described can utilize either nonradioactive hapten-conjugated probes that are detected by enzyme-coupled antibodies, or radioactively labeled probes that are detected by autoradiography. The ability to visualize miRNA expression patterns in tissue sections provides an additional tool for the analyses of miRNA expression and function. In addition, the use of radioactively labeled probes should facilitate quantitative analyses of changes in miRNA gene expression.

Cloning, identification, and expression analysis at the stage of gonadal sex differentiation of chicken miR-363 and 363*

“…Up to now, there were three different ways reported to detect mature miRNAs by ISH. Robert and David described a modified ISH method for miRNAs detection with RNA probes, in combination with specific wash conditions based on tetramethylammonium chloride and RNase A treatment to generate highly sequence specific conditions [27]. While this work was in progress, Obernosterer et al [28] reported another ISH method, which focused on the use of LNA that allowed a significant increase in the hybridization temperature and thereby an enhanced stringency for short probes as required for miRNA detection.…”

Basal microRNA expression patterns in reward circuitry of selectively bred high-responder and low-responder rats vary by brain region and genotype

“…ISH probes were generated from RNA oligonucleotides (Invitrogen, Carlsbad, CA) with minor changes to the labeling reaction as follows: 0.75 l T4 DNA Kinase, 1 l 10ϫ Polynucleotide Kinase Buffer (Affymetrix, Santa Clara, CA), 2 l RNA (20 pmol/l), and 6.25 l 33 P-␥ATP (Perkin Elmer, Waltham, MA) and incubated at 37°C for 30 min (45). Antisense and control (two interior mismatched nucleotides) RNA oligonucleotides (Invitrogen) were synthesized based on sequences obtained from miRBase.org and hybridization specificity as determined through comparative ISHs failing to yield autoradiographic signal above background (Fig.…”

Everything you wanted to know about small RNA but were afraid to ask

“…60,[138][139][140][141][142][143][144] In situ hybridization detection of microRNAs has also been reported by several groups. [145][146][147] It is noteworthy that standard formalin-fixation and paraffin-embedding of tissues seems to preserve microRNAs in a reasonably intact and extractable state. 146,148,149 Once these tools are clinically validated more extensively, they should enable microRNA measurements to become a part of the pathologist's diagnostic armamentarium.…”