Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification

Silahtaroglu, Asli; Nolting, Dorrit; Dyrskjøt, Lars; Berezikov, Eugène; Møller, Mette; Tommerup, Niels; Kauppinen, Sakari

doi:10.1038/nprot.2007.313

Cited by 211 publications

(166 citation statements)

References 22 publications

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“…miRNA in situ hybridization . MiRNA expression was assessed from paraffi n sections as previously described 34 . In brief, aft er 2 h pre-hybridization, a 5 ′ -FITClabeled miRCURY LNA probe targeting miR-23b ( Exiqon ) was hybridized to proteinase K-treated 10 mm sections at 37 ° C for 12 h. Th e images are taken and analysed by ImageXpress Micro ( Molecular Devices ).…”

Section: Methodsmentioning

confidence: 99%

Genome-wide functional screening of miR-23b as a pleiotropic modulator suppressing cancer metastasis

et al. 2011

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miRNA globally deregulates human carcinoma. A critical open question is how many miRNAs functionally participate in cancer development, particularly in metastasis. We systematically evaluate the capability of all known human miRNAs to regulate certain metastasis-relevant cell behaviours. To perform the high-throughput screen of miRNAs, which regulate cell migration, we developed a novel self-assembled cell microarray. Here we show that over 20 % of miRNAs have migratory regulation activity in diverse cell types, indicating a general involvement of miRNAs in migratory regulation. MiR-23b, which is downregulated in human colon cancer samples, potently mediates the multiple steps of metastasis, including tumour growth, invasion and angiogenesis in vivo . It regulates a cohort of prometastatic targets, including FZD7 or MAP3k1 . These fi ndings provide new insight into the physiological and potential therapeutic importance of miRNAs as a new class of functional modulators.

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Section: Methodsmentioning

confidence: 99%

Genome-wide functional screening of miR-23b as a pleiotropic modulator suppressing cancer metastasis

et al. 2011

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“…In fact, this kind of LNA/DNA mixmer is the standard probe design employed for in situ hybridization experiments to detect miRNA species in histology. 33,34 Typically, the probes used in these methods employ a repeating pattern of two DNAs followed by one LNA nucleotide and incorporate seven LNAs in a 21-23mer probe. The relative potency of a variety of AMO designs using a miR-21 reporter system were directly compared by Lennox and Behlke 18 and LNA mixmers were among the most potent designs tested (Table 1).…”

Section: Inhibiting Mirna Function With Synthetic Oligonucleotides Dementioning

confidence: 99%

Chemical modification and design of anti-miRNA oligonucleotides

2011

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Antisense techniques have been employed for over 30 years to suppress expression of target RNAs. Recently, microRNAs (miRNAs) have emerged as a new class of small, non-coding, regulatory RNA molecules that widely impact gene regulation, differentiation and disease states in both plants and animals. Antisense techniques that employ synthetic oligonucleotides have been used to study miRNA function and some of these compounds may have potential as novel drug candidates to intervene in diseases where miRNAs contribute to the underlying pathophysiology. Anti-miRNA oligonucleotides (AMOs) appear to work primarily through a steric blocking mechanism of action; these compounds are synthetic reverse complements that tightly bind and inactivate the miRNA. A variety of chemical modifications can be used to improve the performance and potency of AMOs. In general, modifications that confer nuclease stability and increase binding affinity improve AMO performance. Chemical modifications and/or certain structural features of the AMO may also facilitate invasion into the miRNA-induced silencing complex. In particular, it is essential that the AMO binds with high affinity to the miRNA 'seed region', which spans bases 2-8 from the 5¢-end of the miRNA.

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“…Shi et al, 2011) and methods for labeling enhancement such as gold-silver intensification (e.g. Dobo et al, 2011) and tyramide signal amplification (Kerstens et al, 1995;Silahtaroglu et al, 2007). It is therefore necessary to ask critical questions such as: Discrepancies should raise concerns about antibody specificity.…”

Section: Testing On Tissue Sectionsmentioning

confidence: 99%

Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

Danbolt

Zhou

Furness

et al. 2016

Glia

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Immunocytochemistry and Western blotting are still major methods for protein localization, but they rely on the specificity of the antibodies. Validation of antibody specificity remains challenging mostly because ideal negative controls are often unavailable. Further, immunochemical labeling patterns are also influenced by a number of other factors such as postmortem changes, fixation procedures and blocking agents as well as the general assay conditions (e.g., buffers, temperature, etc.). Western blotting similarly depends on tissue collection and sample preparation as well as the electrophoretic separation, transfer to blotting membranes and the immunochemical probing of immobilized molecules. Publication of inaccurate information on protein distribution has downstream consequences for other researchers because the interpretation of physiological and pharmacological observations depends on information on where ion channels, receptors, enzymes or transporters are located. Despite numerous reports, some of which are strongly worded, erroneous localization data are being published. Here we describe the extent of the problem and illustrate the nature of the pitfalls with examples from studies of neurotransmitter transporters. We explain the importance of supplementing immunochemical observations with other measurements (e.g., mRNA levels and distribution, protein activity, mass spectrometry, electrophysiological recordings, etc.) and why quantitative considerations are integral parts of the quality control. Further, we propose a practical strategy for researchers who plan to embark on a localization study. We also share our thoughts about guidelines for quality control. GLIA 2016;64:2045–2064

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Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification

Cited by 211 publications

References 22 publications

Genome-wide functional screening of miR-23b as a pleiotropic modulator suppressing cancer metastasis

Genome-wide functional screening of miR-23b as a pleiotropic modulator suppressing cancer metastasis

Chemical modification and design of anti-miRNA oligonucleotides

Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

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