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目的观察不同RNA抽提方法对激光显微切割(laser microdissection,LMD)分离牙胚样品总RNA浓度及完整度影响。方法本实验按照RNA抽提的不同方式分为试剂盒组、经典Trizol法组以及改良Trizol法组。以LMD技术获取胚胎17d(embryonic 17,E17)和出生后2 d(postnatal 2,PN2)两时期CD1小鼠下颌第一磨牙的牙胚组织;以3种不同方法提取各组总RNA后再以安捷伦公司的2100生物分析仪所提供的核糖体RNA 28 S/18 S比值(28 S/18 S ratio)、RNA完整度数值(RNA in-tegrity number,RIN),以及样品电泳图的图形来综合判断样品完整度。比较各组RNA的浓度和完整度,以SPSS软件进行统计分析。结果改良Trizol法所获得RNA浓度最高,与其余2组相比,差异均有显著性;经典Trizol法组RNA浓度略高于试剂盒组,但差异并无显著性;3组间以RIN值、28 S/18 S以及样品电泳图的图形为完整度指标进行比较,均无显著性差异。结论3种RNA抽提方式对样品总RNA获得率和完整度的影响,以改良Trizol法提取的总RNA获得率最高,而3种方法对所提取总RNA完整度影响无统计学差异。改良Trizol法将有利于获得符合高通量实验质检标准的芯片实验样本。
Objective To observe the effect of different RNA extraction methods on total RNA concentration and integrity of tooth germ samples isolated by laser microdissection (LMD). Methods According to different ways of RNA extraction, the experiment was divided into kit group, classic Trizol group and modified Trizol group. The tooth germ tissues of the mandibular first molar of embryos 17d (embryonic 17, E17) and postnatal 2 (PN2) were obtained by LMD technique. The total RNA of each group was extracted by three different methods The 28 S / 18 S ratio, the RNA in-tegrity number (RIN), and the pattern of the sample electrophoresis provided by Agilent 2100 Bioanalyzer were used to synthesize Determine the sample integrity. The concentrations and completeness of RNA in each group were compared and analyzed by SPSS software. Results The Trizol method showed the highest concentration of RNA, which was significantly different from the other two groups. The RNA concentration of the classic Trizol group was slightly higher than that of the kit group, but the difference was not significant. The RIN value, 28 S / 18 S and the pattern of the sample electrophoresis for the integrity index were compared, no significant difference. Conclusion The total RNA extraction rate and integrity of three samples were significantly different from those of the other three RNA extraction methods. The highest extraction rate of total RNA was obtained by modified Trizol method. However, no significant difference was found between the three methods. Improved Trizol method will be conducive to obtain high-throughput experimental quality control chip experimental samples.