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目的探讨miRNA原位杂交技术的多种影响因素,以完善miRNA原位杂交分析方法。方法选择不同组织来源、保存时间、切片厚度的常规病理组织切片进行U6探针的原位杂交分析;选择不同浓度的蛋白酶K对组织切片与MDA-MB-231爬片细胞进行U6探针的原位杂交分析;实时定量RT-PCR方法和爬片细胞原位杂交对BT549、T47D细胞株进行miR-375的表达分析。结果不同组织来源、保存时间、切片厚度的石蜡标本均能显示细胞核内的U6表达;组织切片原位杂交实验中,乳腺组织切片20μg/ml的蛋白酶K作用组较其他组的原位杂交效果好;肝脏组织切片200μg/ml的蛋白酶K作用组的显色最好;爬片细胞原位杂交实验中,2μg/ml的蛋白酶K作用组的显色更深;U6与miR-375的原位杂交最适浓度分别为1ng/ml和100ng/ml;实时定量RT-PCR和爬片细胞原位杂交结果均显示,BT-549的miR-375表达明显低于T47D。结论 miRNA原位杂交操作能在常规固定并长期保存的石蜡组织标本上操作;蛋白酶K与探针的浓度应当依据病理标本类型等多因素预实验摸索;细胞水平的实时定量RT-PCR和爬片细胞原位杂交分析结果的比对为确定miRNA探针的特异性提供了帮助。
Objective To explore various factors affecting in situ hybridization of miRNAs to improve the miRNA in situ hybridization assay. Methods U6 probe in situ hybridization was selected by routine histological sections with different tissue sources, storage time and slice thickness. Different concentrations of proteinase K were used to test the tissue sections and MDA-MB-231 cells for U6 probe Bit hybridization analysis; real-time quantitative RT-PCR and in situ hybridization of cells in slices of BT549, T47D miR-375 expression analysis. Results The expression of U6 in the nucleus of the cells was observed in paraffin specimens from different tissues, storage time and slice thickness. In the in situ hybridization of tissue sections, the effect of in situ hybridization with 20μg / ml breast tissue section was better than that of other groups ; Liver tissue sections 200μg / ml protease K role of the best color group; cell slice in situ hybridization experiments, 2μg / ml of proteinase K role group deeper color; U6 and miR-375 in situ hybridization Ml and 100ng / ml, respectively. The results of real-time quantitative RT-PCR and slide cell in situ hybridization showed that the expression of miR-375 in BT-549 was significantly lower than that in T47D. CONCLUSIONS: miRNA in situ hybridization can be performed on routinely fixed and long-term preservation of paraffin-embedded tissue specimens. Proteinase K and probe concentration should be based on multifactorial pre-experiment testing such as pathological specimen types. Real-time quantitative RT-PCR and slide- Alignment of cell-in-situ hybridization results has helped to determine the specificity of the miRNA probe.