Demonstration of microRNA using in situ hybridisation on formalin fixed paraffin wax samples using conventional oligonucleotide probes: A comparison with the use of locked nucleic acid probes

Warford, A., Rahman, N., Riberio, D. and Uysal Onganer, P. 2020. Demonstration of microRNA using in situ hybridisation on formalin fixed paraffin wax samples using conventional oligonucleotide probes: A comparison with the use of locked nucleic acid probes. British Journal of Biomedical Science. 77 (3), pp. 135-141. https://doi.org/10.1080/09674845.2020.1748913

TitleDemonstration of microRNA using in situ hybridisation on formalin fixed paraffin wax samples using conventional oligonucleotide probes: A comparison with the use of locked nucleic acid probes
TypeJournal article
AuthorsWarford, A.
Rahman, N.
Riberio, D.
Uysal Onganer, P.
Abstract

Background: MicroRNAs (miRNA) regulate the translation of mRNA during gene expression and investigations have highlighted their importance in pathophysiology. qRT-PCR is currently the gold standard method for detecting changes in miRNA expression. However, when used on heterogeneous samples, it cannot identify individual cell types harbouring miRNAs. For this, in situ hybridisation (ISH) can be used. ISH methods using locked nucleic acid (LNA) probes have been shown to give reliable results in formalin fixed paraffin embedded (FFPE) samples. In this study their use has been directly compared with conventional oligonucleotide probes (COP) for ISH.

Methods: FFPE samples of colorectal adenocarcinoma, squamous carcinoma of lung and cases of invasive breast carcinoma were used to evaluate COP and LNA methods for the demonstration of miR-126 and miR-205. To demonstrate the utility of the COP method demonstration of miR-21 in 19 Gleason stage 7 prostate biopsy FFPE tissues was also undertaken. The demonstration of miR-21 by ISH in high and low expressing prostate cancer cell lines was also compared with qRT-PCR.

Results: Similar results were obtained using the COP and LNA ISH methods for the demonstration of miR-126 and miR-205. miR-21 was successfully demonstrated in the prostate cancer samples by COP ISH and expression levels of the miRNA demonstrated in the cell lines corresponded with qRT-PCR.

Conclusion: This study has shown that simplification of ISH protocols by the use of COPs provides equivalent results to the use of LNA methods and it can be used to precisely identify cells in which miRNA are expressed.

Keywordsin situ hybridisation, microRNA, oligonucleotide probes, locked nucleic acid probes, prostate cancer.
JournalBritish Journal of Biomedical Science
Journal citation77 (3), pp. 135-141
ISSN0967-4845
Year2020
PublisherTaylor & Francis
Accepted author manuscript
File Access Level
Open (open metadata and files)
Digital Object Identifier (DOI)https://doi.org/10.1080/09674845.2020.1748913
Publication dates
Published online29 Apr 2020

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