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毛细管电泳测定micro RNA346基因多态性的方法研究
The Detection of Micro RNA346 Gene Polymorphism by Capillary Electrophoresis
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摘要: 目的建立micro RNA346基因多态性的毛细管电泳（CE）测定方法。方法采用血液/细胞/组织基因组DNA提取试剂盒提取血清样品基因组DNA，PCR扩增micro RNA346目的基因，BciT130Ⅰ限制性内切酶酶切，产物用CE测定。对CE筛分介质质量浓度、分离电压等参数进行了优化。在优化的条件下（筛分介质质量浓度为10 g/L分离电压为12 kV)，分别测定类风湿性关节炎患者和正常人血清样品micro RNA346酶切产物，并对其进行基因分型。结果在优化的CE实验条件下（筛分介质质量浓度为10 g/L，分离电压为12 kV，25 min内可完成micro RNA346基因酶切产物检测。方法日内相对标准偏差（RSD）为0.43%～0.63%，日间RSD为1.49%～1.56%。用该法测定了96份类风湿性关节炎患者样品和43份正常人样品，结果均为micro RNA346 Ⅰ型，未发现micro RNA346 Ⅱ型。结论本研究建立的方法操作简单，具有高效、快速、样品用量少、自动化程度高等优点，适用于micro RNA类小分子RNA基因多态性的测定。Abstract: Objective To develop a method for the detection of micro RNA346 gene polymorphism by capillary electrophoresis (CE). MethodsThe genome DNA was extracted with the kit of blood/cell/tissue genome DNA extraction, then micro RNA346 gene was amplified by PCR, digested by BciT130Ⅰrestriction enzyme and detected by CE. The conditions for CE separation were optimized. Samples from rheumatoid arthritis patients and healthy persons were detected under the optimal conditions. ResultsUnder the optimized experimental conditions of CE (sieving medium mass concentration was 10 g/L and the separation voltage was 12 kV), the detection of the digested products of microRNA346 gene could be completed within 25 min. The intra-day relative standard deviation (RSD) of the method was 0.43%-0.63% and inter-day RSD was 1.49%-1.56%.Samples from 96 rheumatoid arthritis patients and 43 healthy persons were analyzed by the proposed method. The results showed that only micro RNA346Ⅰtype was detected but micro RNA346 Ⅱ type wasn’t. ConclusionThis method is easy to operate, and has the advantages of high efficiency, fast speed, less sample consumption and high automation level. This method is suitable for the determination of RNA gene polymorphism of mirco RNA.
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