Department of Clinical Immunology, Zealand University Hospital, Koege, Ringstedgade 77B, 4700, Naestved, Denmark.
Department of Science and Environment, Roskilde University, Roskilde, Denmark.
Sci Rep. 2021 Feb 24;11(1):4504. doi: 10.1038/s41598-021-83888-5.
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.
使用地高辛标记的锁核酸（LNA）探针通过原位杂交（ISH）鉴定靶组织 microRNAs（miR）受到分析前参数的影响。为了确定常见 microRNAs 的最佳检索方法，多块组织由石蜡包埋的扁桃体、宫颈、胎盘和增生性前列腺组织组成。组织在 10%甲醛中的固定时间为 5-144 小时（h）。多块组织的切片（5μm）接受预处理程序的组合：蛋白酶 K（PK）消化或热诱导 microRNA 检索（HmiRR）的时间长短不同，使用靶标检索溶液（TRS）pH 6.1 或 9，有无酶处理（胃蛋白酶）。总体分类的结果为：TRS pH 9 与 PK 相比；p=2.9e-23，TRS pH 9 与 TRS pH 6.1 相比；p=1.1e-14，TRS pH 6.1 与 PK 相比；p=2.9e-03。较长的固定时间导致最佳 microRNA 保存和染色强度（长 vs. 短：p=3.5e-47，长 vs. 中：p=1.6e-44，中 vs. 短：p=4.3e-16），使用 HmiRR TRS pH 9 增强，有或没有胃蛋白酶，可提供高灵敏度和特异性。这些观察结果与其他 ISH 技术（例如信使核糖核酸）相冲突，后者通常需要较短的固定时间，因此需要进一步研究。
