MMISH: Multicolor microRNA in situ hybridization for paraffin embedded samples

Lei, Zhiyong; Mil, Alain van; Xiao, Junjie; Metz, Corina H.G.; Eeuwijk, Esther C M van; Doevendans, Pieter A.; Sluijter, Joost P.G.

doi:10.1016/j.btre.2018.e00255

Cited by 11 publications

(16 citation statements)

References 29 publications

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“…Previous studies have used proteinase K for permeabilization, applying various concentrations and incubation times (temperature with enzyme) 7 , 16 , 23 , 25 , 28 . Masuda et al demonstrated that proteinase K was effective for retrieval of RNA even in tissue exposed to a longer fixation time in formalin and in archival samples.…”

Section: Discussionmentioning

confidence: 99%

A novel approach for microRNA in situ hybridization using locked nucleic acid probes

Paulsen

Bzorek

Olsen

et al. 2021

Sci Rep

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Identification of target tissue microRNAs (miR) using in situ hybridization (ISH), with digoxigenin-labeled locked nucleic acid (LNA) probes, is influenced by preanalytic parameters. To determine the best retrieval method for common microRNAs, a multiblock composed of paraffin-embedded tonsil, cervix, placenta, and hyperplastic prostate tissue were included. Tissue were fixed in 10% formalin in a range of 5–144 hours (h). Cut sections (5 μm) from the multiblock were subjected to combinations of pretreatment procedures: variable periods of proteinase K (PK) digestion or Heat-induced microRNA Retrieval (HmiRR) using target retrieval solution (TRS) pH 6.1 or 9, with or without enzymatic treatment (pepsin). Results for the overall categories: TRS pH 9 versus PK; p = 2.9e−23, TRS pH 9 versus TRS pH 6.1; p = 1.1e−14, TRS pH 6.1 versus PK; p = 2.9e−03. A long fixation time, resulted in the best microRNA preservation and staining intensity (long vs. short: p = 3.5e−47, long vs. moderate: p = 1.6e−44, moderate vs. short: p = 4.3e−16), was enhanced using HmiRR TRS pH 9 with or without pepsin providing high sensitivity and specificity. These observations conflict with other ISH techniques (e.g., messenger ribonucleic acid), which typically require shorter fixation periods, and therefore, further studies are warranted.

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Section: Discussionmentioning

confidence: 99%

A novel approach for microRNA in situ hybridization using locked nucleic acid probes

Paulsen

Bzorek

Olsen

et al. 2021

Sci Rep

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“…The detailed protocol for miRNA in situ hybridization has been described previously ( 18 ). Briefly, 10 mm sections were fixed with 4% PFA for 10 min before proteinase K treatment (5 mg/ml) for 10 min.…”

Section: Methodsmentioning

confidence: 99%

miR-132/212 Impairs Cardiomyocytes Contractility in the Failing Heart by Suppressing SERCA2a

Lei

Wahlquist

Azzouzi

et al. 2021

Front. Cardiovasc. Med.

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Compromised cardiac function is a hallmark for heart failure, mostly appearing as decreased contractile capacity due to dysregulated calcium handling. Unfortunately, the underlying mechanism causing impaired calcium handling is still not fully understood. Previously the miR-132/212 family was identified as a regulator of cardiac function in the failing mouse heart, and pharmaceutically inhibition of miR-132 is beneficial for heart failure. In this study, we further investigated the molecular mechanisms of miR-132/212 in modulating cardiomyocyte contractility in the context of the pathological progression of heart failure. We found that upregulated miR-132/212 expressions in all examined hypertrophic heart failure mice models. The overexpression of miR-132/212 prolongs calcium decay in isolated neonatal rat cardiomyocytes, whereas cardiomyocytes isolated from miR-132/212 KO mice display enhanced contractility in comparison to wild type controls. In response to chronic pressure-overload, miR-132/212 KO mice exhibited a blunted deterioration of cardiac function. Using a combination of biochemical approaches and in vitro assays, we confirmed that miR-132/212 regulates SERCA2a by targeting the 3′-end untranslated region of SERCA2a. Additionally, we also confirmed PTEN as a direct target of miR-132/212 and potentially participates in the cardiac response to miR132/212. In end-stage heart failure patients, miR-132/212 is upregulated and correlates with reduced SERCA2a expression. The up-regulation of miR-132/212 in heart failure impairs cardiac contractile function by targeting SERCA2a, suggesting that pharmaceutical inhibition of miR-132/212 might be a promising therapeutic approach to promote cardiac function in heart failure patients.

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“…HCR is commonly used to detect mRNA in all kinds of cells and tissues, or in the whole organism. The third generation of the HCR methodology is establishing which method uses the same principles as other in situ hybridization techniques, but improves the probes and amplifiers involved in the reaction to provide the suppression of background by ensuring that each of the reagents, even if they bind non-specifically, will not amplify the background [48,49]. Reactions use two types of hairpin nucleotides, H1 and H2, which consist of the toehold, stem, and loop domains.…”

Section: In Situ Hybridization Chain Reactionmentioning

confidence: 99%

“…Reactions use two types of hairpin nucleotides, H1 and H2, which consist of the toehold, stem, and loop domains. A visualization of the in situ chain reaction and reaction protocol is in Figure 4 [49]. Domains are metastable and self-assembling when an initiator is not presented.…”

Section: In Situ Hybridization Chain Reactionmentioning

confidence: 99%

Selected In Situ Hybridization Methods: Principles and Application

et al. 2021

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We are experiencing rapid progress in all types of imaging techniques used in the detection of various numbers and types of mutation. In situ hybridization (ISH) is the primary technique for the discovery of mutation agents, which are presented in a variety of cells. The ability of DNA to complementary bind is one of the main principles in every method used in ISH. From the first use of in situ techniques, scientists paid attention to the improvement of the probe design and detection, to enhance the fluorescent signal intensity and inhibition of cross-hybrid presence. This article discusses the individual types and modifications, and is focused on explaining the principles and limitations of ISH division on different types of probes. The article describes a design of probes for individual types of in situ hybridization (ISH), as well as the gradual combination of several laboratory procedures to achieve the highest possible sensitivity and to prevent undesirable events accompanying hybridization. The article also informs about applications of the methodology, in practice and in research, to detect cell to cell communication and principles of gene silencing, process of oncogenesis, and many other unknown processes taking place in organisms at the DNA/RNA level.

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MMISH: Multicolor microRNA in situ hybridization for paraffin embedded samples

Cited by 11 publications

References 29 publications

A novel approach for microRNA in situ hybridization using locked nucleic acid probes

A novel approach for microRNA in situ hybridization using locked nucleic acid probes

miR-132/212 Impairs Cardiomyocytes Contractility in the Failing Heart by Suppressing SERCA2a

Selected In Situ Hybridization Methods: Principles and Application

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