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目的:探求不同乙醇预处理方式是否对激光显微切割(Laser microdissection,LMD)技术分离获得的小鼠牙胚组织总RNA完整度有影响。以提高LMD技术获取样品的完整度,使之满足后续基因芯片等高通量实验对样品的质检要求。方法:本实验按照LMD切割前是否乙醇预处理的方式分为无预处理LMD组、梯度乙醇处理组以及100%乙醇处理组三组。以LMD技术获取胚胎17天(Embryonic 17,E17)和出生后2天(Postnatal 2,PN2)两时期CD1小鼠下颌第一磨牙的牙胚组织;提取各组总RNA,再以核糖体RNA 28s/18s比值、RNA完整度数值(RNA Integrity Number,RIN),以及样品电泳图的图形来综合判断样品完整度。数据进行统计分析。结果:100%乙醇处理组,RIN值最高且与另两组相比差异有显著性,梯度乙醇处理组RIN值与无预处理LMD组相比,并无显著性差异;以28s/18s比值为指标,100%乙醇预处理组高于其它两组,但差异并无显著性;梯度乙醇处理组与无预处理LMD组间差异亦无统计学意义。结论:100%乙醇直接处理方式更适合用于LMD切割样品的防降解预处理,可显著减少切割过程中的组织RNA完整度破坏。该步操作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 investigate whether different ethanol pretreatment methods have effects on the integrity of mouse tooth germ tissue total RNA isolated by laser microdissection (LMD) technique. In order to improve the integrity of the samples obtained by the LMD technology so as to meet the quality inspection requirements of the samples by subsequent high-throughput experiments such as gene chips. Methods: According to the method of ethanol pretreatment before LMD cutting, the experiment was divided into three groups: no pretreatment LMD group, gradient ethanol treatment group and 100% ethanol treatment group. The tooth germ tissues of the mandibular first molars of CD1 mice were obtained by LMD technique at embryonic day 17 (E17) and postnatal 2 (PN2) for two periods. Total RNA was extracted from each group, / 18s ratio, RNA Integrity Number (RIN), and the graph of the sample electrophoresis to determine the completeness of the sample. Data for statistical analysis. Results: The RIN value of 100% ethanol group was the highest and there was significant difference compared with the other two groups. There was no significant difference between the RIN values in the gradient ethanol group and those in the non-pretreated LMD group. The ratio of 28s / 18s was Indicators, 100% ethanol pretreatment group was higher than the other two groups, but the difference was not significant; Gradient ethanol group and no pretreatment LMD group, the difference was not statistically significant. Conclusion: The direct treatment of 100% ethanol is more suitable for the anti-degradation pretreatment of LMD cut samples, which can significantly reduce the tissue integrity damage of RNA during the cutting process. This step will be conducive to obtaining high-throughput experimental quality control chip experimental samples.