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

Abstract 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 tons...

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Autores principales:	Isabella W. Paulsen, Michael Bzorek, Jesper Olsen, Birgitte Grum-Schwensen, Jesper T. Troelsen, Ole B. Pedersen
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/211e5c5c60954dffbab730b608f8181b
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spelling	oai:doaj.org-article:211e5c5c60954dffbab730b608f8181b2021-12-02T13:34:58ZA novel approach for microRNA in situ hybridization using locked nucleic acid probes10.1038/s41598-021-83888-52045-2322https://doaj.org/article/211e5c5c60954dffbab730b608f8181b2021-02-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-83888-5https://doaj.org/toc/2045-2322Abstract 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.Isabella W. PaulsenMichael BzorekJesper OlsenBirgitte Grum-SchwensenJesper T. TroelsenOle B. PedersenNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-14 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Isabella W. Paulsen Michael Bzorek Jesper Olsen Birgitte Grum-Schwensen Jesper T. Troelsen Ole B. Pedersen A novel approach for microRNA in situ hybridization using locked nucleic acid probes
description	Abstract 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.
format	article
author	Isabella W. Paulsen Michael Bzorek Jesper Olsen Birgitte Grum-Schwensen Jesper T. Troelsen Ole B. Pedersen
author_facet	Isabella W. Paulsen Michael Bzorek Jesper Olsen Birgitte Grum-Schwensen Jesper T. Troelsen Ole B. Pedersen
author_sort	Isabella W. Paulsen
title	A novel approach for microRNA in situ hybridization using locked nucleic acid probes
title_short	A novel approach for microRNA in situ hybridization using locked nucleic acid probes
title_full	A novel approach for microRNA in situ hybridization using locked nucleic acid probes
title_fullStr	A novel approach for microRNA in situ hybridization using locked nucleic acid probes
title_full_unstemmed	A novel approach for microRNA in situ hybridization using locked nucleic acid probes
title_sort	novel approach for microrna in situ hybridization using locked nucleic acid probes
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/211e5c5c60954dffbab730b608f8181b
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A novel approach for microRNA in situ hybridization using locked nucleic acid probes

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