microRNA原位杂交技术影响因素的探讨  

Discussion on influence factors of microRNA in situ hybridization

作　　者：刘卓琦[1] 杨晓红[1] 肖影群[2] 黄波[2] 章萍[2] 杨仙荷[2] 黄昀[3] 王炜东 王蒙蒙[1] 罗达亚[1] 

机构地区：[1]南昌大学基础医学院生物化学与分子生物学教研室,江西南昌330006 [2]南昌大学附属感染病医院病理科,江西南昌330002 [3]南昌大学第二临床医学院,2011级江西南昌330006

出　　处：《中国组织化学与细胞化学杂志》2014年第6期491-496,共6页Chinese Journal of Histochemistry and Cytochemistry

基　　金：国家自然科学基金项目资助(30860319;81160248;81360313);江西省自然科学青年基金资助(20142BAB215051);江西省研究生创新专项资金资助(YC2013-S006)

摘　　要：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 investigate the effect of various factors on miRNA in situ hybridization technique to improve the analysis method.Methods Routinely handled pathological slices of different tissues,preservation times and slice thicknesses were analyzed for U6 expression with in situ hybridization analysis.In situ hybridization analysis with different concentrations of proteinase K and probes were performed on tissue sections and MDA-MB-231 cell climbing slices,respectively,for miR-375 and U6expressions.The miR-375 expression for 2breast cancer cell lines,BT549 and T47D,were detected with quantitative RT-PCR and in situ hybridization analysis,respectively.Results Paraffin embedded specimens of different tissues,preservation times and slice thicknesses clearly showed the staining of nuclear U6.With in situ hybridization analysis,the group with proteinase K of 20μg/ml had a better result than other groups on tissue sections for miR-375 expression,while the 2μg/ml proteinase K treatment group had a better result on MDAMB-231 cell climbing slices for U6 expression,and the optimum concentrations for U6 and miR-375 were 1ng/ml and 100ng/ml.Both quantitative RT-PCR and in situ hybridization on climbing slices of 2breast cancer cell lines showed that the miR-375 expression of BT-549 was significantly lower than that of T47 D.Conclusion The miRNA in situ hybridization analysis can be conducted on routinely handled formalin-fixed and paraffin-embedded tissue specimens of long-term preservation.Concentrations of proteinaseK andmiRNA probe should be determined with pre-experiments,based on multi-factors such as sample source.Results comparison between quantitative RT-PCR and in situ hybridization on cell climbing slices is helpful to determine the specificity of the miRNA probe.

关 键 词：MICRORNA 原位杂交技术 影响因素 

分 类 号：R36-33[医药卫生—病理学]