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 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.

miRNAs in pancreatic cancer progression and metastasis

“…Due to the intrinsic cellular heterogeneity of many solid tumours and to overcome the limitation of bulk tissue sequencing, spatial Predicts miRNA targets based on a machine-learning algorithm, TarPmiR Allows searches on the interactions between genes in signalling pathways or biological processes in diseases [172] miRDB Predicts miRNA targets by a bioinformatic tool, MirTarget [173] miRTarBase Stores published studies that functionally validated miRNA targets [174] miRCancerdb Stores information on miRNA to gene/protein expression association in cancer, based on TCGA and TargetScan data [175] MiREDiBASE Provides information on miRNA editing and modification that may affect miRNA biogenesis [176] detection of miRNA expressions has recently received a lot of attention to being to investigate and map where a miRNA of interest is localised within a specific tissue. Traditionally this has be achieved via fluorescent in situ hybridisation on formalin fixed paraffin embedded (FFPE) samples [177]. However recent advancements in miRNAscope technology now allows co-visualisation of miRNA, RNA and proteins simultaneously [178].…”

“…Due to the intrinsic cellular heterogeneity of many solid tumours and to overcome the limitation of bulk tissue sequencing, spatial detection of miRNA expressions has recently received a lot of attention to being to investigate and map where a miRNA of interest is localised within a specific tissue. Traditionally this has be achieved via fluorescent in situ hybridisation on formalin fixed paraffin embedded (FFPE) samples [ 177 ]. However recent advancements in miRNAscope technology now allows co-visualisation of miRNA, RNA and proteins simultaneously [ 178 ].…”

Lupus nephritis: The regulatory interplay between epigenetic and MicroRNAs

“…Furthermore, miRNAs can be detected in human cell lines and bodily fluids, such as blood, urine, and cerebrospinal fluid, and miRNA signatures unique to various disorders have been discovered in tissues and bodily fluids (Weber et al, 2010). Using quantitative polymerase chain reaction (qPCR), fluorescence in situ hybridization (FISH), or next-generation sequencing (NGS), differentially expressed miRNAs can be profiled in a variety of specimens (Cao et al, 2021;Paulsen et al, 2021). Previous studies have shed light on the epigenetic mechanisms that play a role in many illnesses and could lead to the development of new diagnostic and therapeutic tools.…”