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

Isabella W Paulsen,Michael Bzorek,Ole B Pedersen,Birgitte Grum-Schwensen,Jesper T Troelsen,Jesper Olsen

doi:10.1038/s41598-021-83888-5

Isabella W Paulsen, Michael Bzorek + Show 4 more

Open Access

https://doi.org/10.1038/s41598-021-83888-5

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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.

Highlights

Over the past decade, noncoding RNA, including microRNAs, has become a hot research topic[1,2,3]
Several studies have shown that the appropriate buffers at high temperatures can be used to recover DNA/RNA12,13, but only few reports support the use of Heat-induced microRNA Retrieval with locked nucleic acid (LNA) microRNA probes and ISH14–16
The present study provides a “pretreatment test battery” model inspired by Taylor et al.[17] for optimization of microRNA retrieval, with the aim of improving microRNA in situ hybridization (ISH) assays of formalin-fixed paraffin-embedded (FFPE) tissue using specific LNA probes[18]

Summary

Introduction

Over the past decade, noncoding RNA (ncRNA), including microRNAs, has become a hot research topic[1,2,3]. For target validation of a given microRNA, tools such as luciferase reporter assays and northern blotting can be used for cell cultures. These methods only reveal the microRNA profile of the whole s ample[4,5] and not where or from which cells the detected microRNAs originate. The present study provides a “pretreatment test battery” model inspired by Taylor et al.[17] for optimization of microRNA retrieval, with the aim of improving microRNA ISH assays of FFPE tissue using specific LNA probes[18]. We compared the morphology and signal intensity across tissues

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