RAKE and LNA-ISH reveal microRNA expression and localization in archival human brain

Nelson, Peter T.; Baldwin, Don A.; Kloosterman, Wigard P.; Kauppinen, Sakari; Plasterk, Ronald H.A.; Mourelatos, Zissimos P.

doi:10.1261/rna.2258506

Cited by 277 publications

(227 citation statements)

References 37 publications

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“…16,17 Other predictions (eg nodes #1 and #6 in Rosenfeld et al 26 ) were supported by qRT-PCR 24 and in situ hybridization. 35,37 Here, we translated this framework into a standardized qRT-PCR test for clinical application. We describe an additional discovery phase, the translation of the results to a qRT-PCR platform, the development of the diagnostic test, and its validation in a clinical laboratory.…”

Section: Discussionmentioning

confidence: 99%

Validation of a microRNA-based qRT-PCR test for accurate identification of tumor tissue origin

et al. 2010

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Identification of the tissue of origin of a tumor is vital to its management. Previous studies showed tissuespecific expression patterns of microRNA and suggested that microRNA profiling would be useful in addressing this diagnostic challenge. MicroRNAs are well preserved in formalin-fixed, paraffin-embedded (FFPE) samples, further supporting this approach. To develop a standardized assay for identification of the tissue origin of FFPE tumor samples, we used microarray data from 504 tumor samples to select a shortlist of 104 microRNA biomarker candidates. These 104 microRNAs were profiled by proprietary quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) on 356 FFPE tumor samples. A total of 48 microRNAs were chosen from this list of candidates and used to train a classifier. We developed a clinical test for the identification of the tumor tissue of origin based on a standardized protocol and defined the classification criteria. The test measures expression levels of 48 microRNAs by qRT-PCR, and predicts the tissue of origin among 25 possible classes, corresponding to 17 distinct tissues and organs. The biologically motivated classifier combines the predictions generated by a binary decision tree and K-nearest neighbors (KNN). The classifier was validated on an independent, blinded set of 204 FFPE tumor samples, including nearly 100 metastatic tumor samples. The test predictions correctly identified the reference diagnosis in 85% of the cases. In 66% of the cases the two algorithm predictions (tree and KNN) agreed on a single-tissue origin, which was identical to the reference diagnosis in 90% of cases. Thus, a qRT-PCR test based on the expression profile of 48 tissue-specific microRNAs allows accurate identification of the tumor tissue of origin.

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Section: Discussionmentioning

confidence: 99%

Validation of a microRNA-based qRT-PCR test for accurate identification of tumor tissue origin

et al. 2010

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“…miR-124 is present in the developing cerebral cortex [25,26,28]. To further investigate the role of miR-124 during neuronal differentiation, we cotransfected a GFP expression vector with miR124 expression vectors or miR-124 2'-O-Me oligos into cortical progenitors isolated from E14.5 mouse embryos and allowed the cortical cells to differentiate in vitro.…”

Section: Mir-124 Alters Neurite Outgrowth In Primary Cortical Neuronsmentioning

confidence: 99%

“…miRNAs also appear to have important roles in the mammalian central nervous system (CNS). Numerous miRNAs are expressed in the CNS [22][23][24], and many neural miRNAs are expressed in spatial and/or temporal patterns that suggest roles in the regulation of CNS development [25][26][27][28][29][30]. MiRNA miR-132 has been shown to regulate neuronal morphogenesis through decreasing levels of GTPase-activating protein, p250GAP [31] and (along with miR-219) has been implicated in the regulation of circadian rhythms [32].…”

Section: Introductionmentioning

confidence: 99%

“…MiR-134 has been shown to regulate dendritic spine development by inhibiting expression of the Limk1 protein kinase [34]. miR-124 is one of the most abundantly expressed miRNAs in the nervous system, being widely expressed in neurons in the brain, retina, and spinal cord [22,25,26,28,[35][36][37]. There are three miR-124 genes, miR-124-1, miR-124-2, and miR-124-3, located on three different chromosomes in mouse and human genomes [38].…”

Section: Introductionmentioning

confidence: 99%

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MicroRNA miR-124 regulates neurite outgrowth during neuronal differentiation

Chung

Deo

et al. 2008

Experimental Cell Research

292

211

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MicroRNAs (miRNAs) are small RNAs with diverse regulatory roles. The miR-124 miRNA is expressed in neurons in the developing and adult nervous system. Here we show that overexpression of miR-124 in differentiating mouse P19 cells promotes neurite outgrowth, while blocking miR-124 function delays neurite outgrowth and decreases acetylated α-tubulin. Altered neurite outgrowth also was observed in mouse primary cortical neurons when miR-124 expression was increased, or when miR-124 function was blocked. In uncommitted P19 cells, miR-124 expression led to disruption of actin filaments and stabilization of microtubules. Expression of miR-124 also decreased Cdc42 protein and affected the subcellular localization of Rac1, suggesting that miR-124 may act in part via alterations to members of the Rho GTPase family. Furthermore, constitutively active Cdc42 or Rac1 attenuated neurite outgrowth promoted by miR-124. To obtain a broader perspective, we identified mRNAs downregulated by miR-124 in P19 cells using microarrays. mRNAs for proteins involved in cytoskeletal regulation were enriched among mRNAs downregulated by miR-124. A miR-124 variant with an additional 5' base failed to promote neurite outgrowth and downregulated substantially different mRNAs. These results indicate that miR-124 contributes to the control of neurite outgrowth during neuronal differentiation, possibly by regulation of the cytoskeleton.

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“…Plasterk and colleagues have used an ISH method based on Locked Nucleic Acid (LNA) oligonucleotide probes to detect mature miRNAs in whole mounts of zebrafish and mouse embryos [ 11,12 ]. The LNAbased ISH methodology has been applied to whole-mount embryos from a variety of vertebrate species, including chicken and medaka [ 13,14 ], and for the analysis of adult human brain sections [ 15 ] as well as mouse tissue sections [ 16,17 ]. Independently, we have developed a miRNA ISH method based upon RNA oligonucleotide probes and have used this method for the detection of mature miRNAs in tissue sections from embryonic and adult mice [ 18 ], as well as from rat and human brains (this article) and zebrafish (our unpublished observations).…”

Section: Introductionmentioning

confidence: 99%

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

Thompson

Deo

Turner

2007

Methods

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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.

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RAKE and LNA-ISH reveal microRNA expression and localization in archival human brain

Cited by 277 publications

References 37 publications

Validation of a microRNA-based qRT-PCR test for accurate identification of tumor tissue origin

Validation of a microRNA-based qRT-PCR test for accurate identification of tumor tissue origin

MicroRNA miR-124 regulates neurite outgrowth during neuronal differentiation

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

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