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

Isabella W. Paulsen, Jesper Thorvald Troelsen, Ole Birger Pedersen, Birgitte Grum-Schwensen, Jesper Olsen, Micheal Bzorek

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

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.
OriginalsprogEngelsk
Artikelnummer4504
TidsskriftScientific Reports
Vol/bind2021
Udgave nummer11
ISSN2045-2322
DOI
StatusUdgivet - 24 feb. 2021

Emneord

  • microRNA, in situ hybridization

Citer dette