MicroRNA In Situ Hybridization in Tissue Microarrays

Turnock-Jones, Julia J.; Quesne, John Le

doi:10.1007/978-1-4939-1459-3_8

Cited by 6 publications

(7 citation statements)

References 8 publications

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“…Indeed, of these 406 sequences, 115 are offset at the 5′ end with respect to the miRBase entries. This changes the seed sequence and hence possibly affects any type of detection system based on hybridization [e.g., RT-qPCR (12), Luminex (14), microarray (94), northern blotting (149) or in situ hybridization, 148], albeit to a different extent according to the method. It may also affect the utility of methods used to identify potential targets (e.g., miR-CLIP; 71) or those that mimic or target miRNAs (6, 15, 16, 92, 115).…”

Section: The Establishment Of a Microrna Gene Database: Mirgenedborgmentioning

confidence: 99%

A Uniform System for the Annotation of Vertebrate microRNA Genes and the Evolution of the Human microRNAome

Fromm¹,

et al. 2015

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Although microRNAs (miRNAs) are among the most intensively studied molecules of the past 20 years, determining what is and what is not a miRNA has not been straightforward. Here, we present a uniform system for the annotation and nomenclature of miRNA genes. We show that fewer than a third of the 1,881 human miRBase entries, and only approximately 16% of the 7,095 metazoan miRBase entries, are robustly supported as miRNA genes. Furthermore, we show that the human repertoire of miRNAs has been shaped by periods of intense miRNA innovation, and that mature gene products show a very different tempo and mode of sequence evolution than star products. We establish a new open access database -- MirGeneDB (http://mirgenedb.org) -- to catalog this set of robustly supported miRNAs, which complements the efforts of miRBase, but differs from it by annotating the mature versus star products, and by imposing an evolutionary hierarchy upon this curated and consistently named repertoire.

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Section: The Establishment Of a Microrna Gene Database: Mirgenedborgmentioning

confidence: 99%

A Uniform System for the Annotation of Vertebrate microRNA Genes and the Evolution of the Human microRNAome

Fromm¹,

et al. 2015

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“…The concept is straightforward and in general similar to the traditional in situ hybridizations for long coding mRNAs: a pre-labeled nucleic acid sequence, called probe, complementary to the selected miRNA is used to visualize the localization of the specific miRNA. Based on these well-established mRNA in situ procedures, several protocols have been developed to detect miRNA expression, which have advanced our understanding of how and where miRNAs are located [ [12] , [13] , [14] , [15] , [16] , [17] , [18] ].…”

Section: Introductionmentioning

confidence: 99%

“…However, one major drawback of EDC fixation is that it destroys the epitope of cell surface markers, which makes it difficult to perform subsequent immunohistochemical staining [ 18 ]. Still, to date only highly abundant miRNAs have been localized and defined by in situ hybridization [ [12] , [13] , [14] , [15] , [16] , [17] , [18] ], suggesting still low sensitivity of this technique and the need for further optimization. Moreover, most of the available protocols require cryopreserved tissue samples [ 12 , 13 ], while most clinical grade patient samples are paraffin embedded to better preserve morphology.…”

Section: Introductionmentioning

confidence: 99%

MMISH: Multicolor microRNA in situ hybridization for paraffin embedded samples

Lei

Mil

Xiao

et al. 2018

Biotechnology Reports

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HighlightsA robust, sensitive and flexible multicolor miRNA in situ hybridization (MMISH) technique for paraffin embedded sections can be combined with both immunohistochemical and immunofluorescent staining.Usage of urea in our buffers which enhances the target-probe affinity by preventing intermolecular interaction within miRNAs or individual probes, and by reversing the EDC fixation induced epitope loss by denaturing the antigens, less toxic compared to toxic formamide.Second, it can be combined with immunofluorescent stainings, which allows one to analyze the expression and precise (sub)cellular location of the miRNA of interest.

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“…, Saponin, remove cholesterol from membranes in highly selective ways, but widely used Triton X-100 is not selective, which can lead to elimination of both proteins and lipids [ 63 ]. To reduce cellular RNA diffusion, treatment with proteinases and detergents was either limited or eliminated in some miRNA ISH studies [ 22 , 64 ]. The need for additional permeabilization decreases with the use of EDC fixation due to its auxiliary permeabilization activity.…”

Section: Approaches For Mirna Ishmentioning

confidence: 99%

“…This technique enables fast detection of specific miRNAs in many tissue samples simultaneously. This high throughput analysis was performed with LNA/DNA probes and TSA labeling [ 64 ]. Automation in multiplex miRNA detection was also described with the use of the same detection system [ 69 ].…”

Section: Applications Of Small Rna Ishmentioning

confidence: 99%

Small RNA Detection by in Situ Hybridization Methods

Urbanek

Nawrocka

Krzyżosiak

2015

IJMS

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Small noncoding RNAs perform multiple regulatory functions in cells, and their exogenous mimics are widely used in research and experimental therapies to interfere with target gene expression. MicroRNAs (miRNAs) are the most thoroughly investigated representatives of the small RNA family, which includes short interfering RNAs (siRNAs), PIWI-associated RNA (piRNAs), and others. Numerous methods have been adopted for the detection and characterization of small RNAs, which is challenging due to their short length and low level of expression. These include molecular biology methods such as real-time RT-PCR, northern blotting, hybridization to microarrays, cloning and sequencing, as well as single cell miRNA detection by microscopy with in situ hybridization (ISH). In this review, we focus on the ISH method, including its fluorescent version (FISH), and we present recent methodological advances that facilitated its successful adaptation for small RNA detection. We discuss relevant technical aspects as well as the advantages and limitations of ISH. We also refer to numerous applications of small RNA ISH in basic research and molecular diagnostics.

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MicroRNA In Situ Hybridization in Tissue Microarrays

Cited by 6 publications

References 8 publications

A Uniform System for the Annotation of Vertebrate microRNA Genes and the Evolution of the Human microRNAome

A Uniform System for the Annotation of Vertebrate microRNA Genes and the Evolution of the Human microRNAome

MMISH: Multicolor microRNA in situ hybridization for paraffin embedded samples

Small RNA Detection by in Situ Hybridization Methods

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