A Sensitive Alternative for MicroRNA In Situ Hybridizations Using Probes of 2′-O-Methyl RNA + LNA

Søe, Martin Jensen; Møller, Trine; Dufva, Martin; Holmstrøm, Kim

doi:10.1369/0022155411409411

Cited by 55 publications

(45 citation statements)

References 28 publications

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“…In line with the expression of pri-miR-9-6, we could detect mature miR-9 exclusively in RG NSCs (Figure 1C). To confirm the specificity of the signal, we performed additional ISH using two control LNA probes (a scrambled control probe and a probe antisense to miR-9-3p) and two alternative LNA-modified 2′-O-Me RNA oligo probes (Søe et al., 2011) (one control and one miR-9-specific probe). Altogether, these controls demonstrate both the specificity of our miR-9 probe and the stringency of our ISH protocol (Figures S1D and S1E) (LNA probes and their sequences are listed in Table S2).…”

Section: Resultsmentioning

confidence: 99%

A Nuclear Role for miR-9 and Argonaute Proteins in Balancing Quiescent and Activated Neural Stem Cell States

et al. 2016

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SummaryThroughout life, adult neural stem cells (NSCs) produce new neurons and glia that contribute to crucial brain functions. Quiescence is an essential protective feature of adult NSCs; however, the establishment and maintenance of this state remain poorly understood. We demonstrate that in the adult zebrafish pallium, the brain-enriched miR-9 is expressed exclusively in a subset of quiescent NSCs, highlighting a heterogeneity within these cells, and is necessary to maintain NSC quiescence. Strikingly, miR-9, along with Argonaute proteins (Agos), is localized to the nucleus of quiescent NSCs, and manipulating their nuclear/cytoplasmic ratio impacts quiescence. Mechanistically, miR-9 permits efficient Notch signaling to promote quiescence, and we identify the RISC protein TNRC6 as a mediator of miR-9/Agos nuclear localization in vivo. We propose a conserved non-canonical role for nuclear miR-9/Agos in controlling the balance between NSC quiescence and activation, a key step in maintaining adult germinal pools.

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Section: Resultsmentioning

confidence: 99%

A Nuclear Role for miR-9 and Argonaute Proteins in Balancing Quiescent and Activated Neural Stem Cell States

et al. 2016

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“…Formaldehyde cross-links are reversible and may cause some miRNA release during processing. Dual fixation with formaldehyde and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) leads to increased miRNA retention and enhanced sensitivity (Pena et al, 2009), though not all studies have replicated this effect (Jørgensen et al, 2010; Lu and Tsourkas, 2009; Søe et al, 2011). …”

Section: Functional Validation Of Putative Mirna-transcript Interactionsmentioning

confidence: 99%

Advances in microRNA experimental approaches to study physiological regulation of gene products implicated in CNS disorders

Long

Lahiri

2012

Experimental Neurology

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The central nervous system (CNS) is a remarkably complex organ system, requiring an equally complex network of molecular pathways controlling the multitude of diverse, cellular activities. Gene expression is a critical node at which regulatory control of molecular networks is implemented. As such, elucidating the various mechanisms employed in the physiological regulation of gene expression in the CNS is important both for establishing a reference for comparison to the diseased state and for expanding the set of validated drug targets available for disease intervention. MicroRNAs (miRNAs) are an abundant class of small RNA that mediates potent inhibitory effects on global gene expression. Recent advances have been made in methods employed to study the contribution of these miRNAs to gene expression. Here we review these latest advances and present a methodological workflow from the perspective of an investigator studying the physiological regulation of a gene of interest. We discuss methods for identifying putative miRNA target sites in a transcript of interest, strategies for validating predicted target sites, assays for detecting miRNA expression, and approaches for disrupting endogenous miRNA function. We consider both advantages and limitations, highlighting certain caveats that inform the suitability of a given method for a specific application. Through careful implementation of the appropriate methodologies discussed herein, we are optimistic that important discoveries related to miRNA participation in CNS physiology and dysfunction are on the horizon.

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“…Several methods have been described for in situ hybridization of miRNAs in tissue sections (Chen, 2004; Deo et al, 2006; Nelson et al, 2006; Ryan et al, 2006; Obernosterer et al, 2007; Schaefer et al, 2007; Sempere et al, 2007; Silahtaroglu et al, 2007; Thompson et al, 2007; Williams et al, 2007; Bak et al, 2008; Nuovo, 2008; Wang et al, 2008; Lu and Tsourkas, 2009; Nelson and Wilfred, 2009; Pena et al, 2009; Yamamichi et al, 2009; Havelda, 2010; Jorgensen et al, 2010; Song et al, 2010; Soe et al, 2011; Shi et al, 2012) and a some for concurrent detection of proteins using IHC or IF (Zaidi et al, 2000; Nuovo et al, 2009; de Planell-Saguer et al, 2010; Nuovo, 2010; Sempere et al, 2010; Schneider et al, 2011; Wu et al, 2011; Herzer et al, 2012; Nielsen and Holmstrom, 2013; Sempere and Korc, 2013). Among the published methods for co-detection of miRNA and proteins in FFPE sections, Nuovo et al described a method for colorimetric in situ hybridization for miRNAs using digoxigenin-labeled LNA probe (Nuovo et al, 2009).…”

Section: Resultsmentioning

confidence: 99%

Combined fluorescent in situ hybridization for detection of microRNAs and immunofluorescent labeling for cell-type markers

Chaudhuri¹,

Yelamanchili²,

Fox³

2013

Front. Cell. Neurosci.

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Identification of the cell type of origin for normal or aberrant gene expression is critical for many studies, and poses a significant problem for some regulatory RNAs such as microRNAs. MicroRNAs are small non-coding RNAs that regulate cellular function by targeting specific mRNAs and reducing the level of their protein product. Aberrant expression of miRNAs in cell-types where they are not normally expressed occurs in several disease conditions. Therefore, it is important to determine not only the expression level of microRNAs, but also where they are expressed. Here we describe a detailed method for fluorescent in situ hybridization (FISH) combined with immunofluorescent labeling for cell-type markers in formalin fixed paraffin embedded (FFPE) sections along with modifications required to adapt the protocol for primary neurons grown in culture. We have combined the specificity and stability of locked nucleic acid (LNA) probes with tyramide signal amplification. To prevent loss of small RNA species, we performed post-fixation with ethylcarbodiimide (EDC). Additionally by omitting protease digestion and using only high temperature with sodium citrate buffer for FFPE sections, we were able to perform immunolabeling for proteins concurrently with in situ hybridization without compromising efficacy of either procedure.

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A Sensitive Alternative for MicroRNA In Situ Hybridizations Using Probes of 2′-O-Methyl RNA + LNA

Cited by 55 publications

References 28 publications

A Nuclear Role for miR-9 and Argonaute Proteins in Balancing Quiescent and Activated Neural Stem Cell States

A Nuclear Role for miR-9 and Argonaute Proteins in Balancing Quiescent and Activated Neural Stem Cell States

Advances in microRNA experimental approaches to study physiological regulation of gene products implicated in CNS disorders

Combined fluorescent in situ hybridization for detection of microRNAs and immunofluorescent labeling for cell-type markers

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