In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes

Kloosterman, Wigard P.; Wienholds, Erno; Bruijn, Ewart de; Kauppinen, Sakari; Plasterk, Ronald H.A.

doi:10.1038/nmeth843

Cited by 564 publications

(450 citation statements)

References 12 publications

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“…This analysis should allow us to follow miRNA expression profiles within epithelial cells that are differently stimulated. Alternatively, it was recently described that locked nucleic acid (LNA)-modified DNA probes could be used to follow the miRNA expression profile in mouse embryos by in situ hybridisation [29]. Such a strategy could reveal the precise location of miRNA within the mammary tissue at various developmental stages.…”

Section: Mirna Expression In Mammary Epithelial Cellsmentioning

confidence: 99%

MicroRNA involvement in mammary gland development and breast cancer

et al. 2006

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-MicroRNA (miRNA) are small non-coding RNA that post-transcriptionally regulate gene expression. In humans, miRNA genes may account for 2 to 3% of the total number of genes. Although the biological functions of most miRNA are unknown, their importance for development, cell proliferation, cell death, and morphogenesis has been demonstrated in several species. One could thus speculate that miRNA should be involved in the regulation of one of the organs that can undergo cycles of cell division, differentiation and dedifferentiation in the adult, the mammary gland. In this paper we summarise several reports dealing with the potential implication of miRNA in the mammary gland, most of them focussed on pathological situations, such as the appearance of breast cancer. These data suggest an implication of miRNA on mammary gland biology. However, direct evidence of this is still lacking. Expression profile analysis of miRNA during the normal mammary gland development could help in addressing this question and in identifying miRNA potentially involved. To this aim, we undertook such an analysis on mouse mammary gland at different stages (virgin, pregnancy, lactation and involution) and will present our preliminary results. gene regulation / mouse / RNA interference / mammary tissue

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Section: Mirna Expression In Mammary Epithelial Cellsmentioning

confidence: 99%

MicroRNA involvement in mammary gland development and breast cancer

et al. 2006

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“…Although powerful, this method requires transgenic animals to visualize expression in vivo. Recently, it has been reported that nonradioactive in situ hybridization with locked nucleic acid (LNA) probes labeled with a digoxigenin hapten can be used to detect miRNAs (Sokol and Ambros, 2005;Wienholds et al, 2005;Kloosterman et al, 2006;Nelson et al, 2006). We have independently developed an in situ hybridization method for miRNA detection based on RNA oligonucleotide probes linked to a fluorescein hapten.…”

Section: Introductionmentioning

confidence: 99%

Detection of mammalian microRNA expression by in situ hybridization with RNA oligonucleotides

Deo

Chung

et al. 2006

Developmental Dynamics

265

216

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We have developed an in situ hybridization procedure for the detection of microRNAs (miRNAs) in tissue sections from mouse embryos and adult organs. The method uses highly specific washing conditions for RNA oligonucleotide probes conjugated to a fluorescein hapten. We show that this method detects predominantly mature miRNAs rather than the miRNA precursors or primary transcripts. We have determined expression patterns for several miRNAs expressed in the developing and adult nervous system, including miR-124a, miR-9, miR-92, and miR-204. Whereas miR-124a is expressed in neurons, miR-9 is expressed in neural progenitors and some neurons, and miR-204 is expressed in the choroid plexus, retinal pigment epithelium, and ciliary body. miR-204 is located in an intron of the TRPM3 gene, and the TRPM3 mRNA is coexpressed with miR-204 in the choroid plexus. We also find that primary transcripts for miR-124a and miR-9 genes are expressed in patterns similar to their respective mature miRNAs. The ability to visualize expression of specific miRNAs in embryos and tissues should aid studies on miRNA function.

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“…Recent advances have shown that miRNA expression during development is highly tissue-specific (9)(10)(11)(12), which suggests that miRNA may be involved in specifying and maintaining tissue identity. For example, the expression of miR-124a is restricted to the brain and spinal cord in the fish and the mouse, and to the ventral nerve cord in the fly (13).…”

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confidence: 99%

MicroRNA‐124a is a key regulator of proliferation and monocyte chemoattractant protein 1 secretion in fibroblast‐like synoviocytes from patients with rheumatoid arthritis

Nakamachi¹,

Kawano²,

Takenokuchi³

et al. 2009

Arthritis & Rheumatism

301

209

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Objective. To elucidate the role of microRNA (miRNA) in the pathogenesis of rheumatoid arthritis (RA), we analyzed synoviocytes from RA patients for their miRNA expression.Methods. Synoviocytes derived from surgical specimens obtained from RA patients were compared with those obtained from osteoarthritis (OA) patients for their expression of a panel of 156 miRNA with quantitative stem-loop reverse transcription-polymerase chain reaction. The miRNA whose expression decreased or increased in RA synoviocytes as compared with OA synoviocytes were identified, and their target genes were predicted by computer analysis. We used an in vitro system of enhancing the expression of specific miRNA by transfection of precursors into synoviocytes, and then we performed proliferation, cell cycle, and apoptosis assays, as well as enzyme-linked immunosorbent assays for cytokine production. The effects of transfection on predicted target protein and messenger RNA (mRNA) were then examined by Western blot analysis and luciferase reporter assay.Results. We found that miR-124a levels significantly decreased in RA synoviocytes as compared with OA synoviocytes. Transfection of precursor miR-124a into RA synoviocytes significantly suppressed their proliferation and arrested the cell cycle at the G 1 phase. We identified a putative consensus site for miR-124a binding in the 3 -untranslated region of cyclin-dependent kinase 2 (CDK-2) and monocyte chemoattractant protein 1 (MCP-1) mRNA. Induction of miR-124a in RA synoviocytes significantly suppressed the production of the CDK-2 and MCP-1 proteins. Luciferase reporter assay demonstrated that miR-124a specifically suppressed the reporter activity driven by the 3 -untranslated regions of CDK-2 and MCP-1 mRNA.Conclusion. The results of this study suggest that miR-124a is a key miRNA in the posttranscriptional regulatory mechanisms of RA synoviocytes.MicroRNA (miRNA) are a well-established class of small (ϳ22 nucleotides) endogenous noncoding RNAs that influence the stability and translation of messenger RNA (mRNA) (1). Using various computational and experimental approaches, hundreds of miRNA have been identified in numerous animal species. The miRNA genes are transcribed by RNA polymerase II as primary miRNA (pri-miRNA) (2,3). The RNase III enzyme Drosha then processes the nuclear pri-miRNA, yielding a ϳ70-nucleotide molecule known as precursor miRNA (pre-miRNA) (4), which is exported from the nucleus. Maturation of the pre-miRNA into miRNA is then mediated by the cytoplasmic enzyme Dicer (5), after which the mature miRNA is loaded into the RNA-induced silencing complex (RISC)

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In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes

Cited by 564 publications

References 12 publications

MicroRNA involvement in mammary gland development and breast cancer

MicroRNA involvement in mammary gland development and breast cancer

Detection of mammalian microRNA expression by in situ hybridization with RNA oligonucleotides

MicroRNA‐124a is a key regulator of proliferation and monocyte chemoattractant protein 1 secretion in fibroblast‐like synoviocytes from patients with rheumatoid arthritis

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