不同乙醇预处理方式对LMD分离牙胚组织总RNA完整度的影响

The effects of different ethanol pretreatment methods on the integrity of total RNA samples of murine tooth germ tissues dissected by LMD system

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 could improve the total RNA integrity of samples dissected by LMD system from murine tooth germ.We try to explore a relatively simple pretreatment method to improve integrity of LMD samples,which can meet the high quality requirements of subsequent microarray experiment.Methods： According to the different ethanol pretreatment methods before cutting,the samples were divided into three groups： LMD group without pretreatment,gradient ethanol pretreatment group and 100% ethanol pretreatment group.The tooth germ tissue of the first lower molar on E17 and PN2 stages were dissected by LMD6000 system.To ensure the purity and the integrity of RNA samples,quality check was adopted by Bioanalyzer 2100 System,Agilent.The 28s/18s Ratio of ribosome RNA,RNA Integrity Number（RIN） and the peak pattern of the electrophoretogram were integrated as the index of RNA integrity.The RNA integrity of all groups were compared by SPSS statistical analysis software.Results： The RIN value of 100% ethanol pretreatment group was the highest and has significant difference compared with another two groups.The comparison between the rest of two groups,the gradient ethanol pretreatment group and LMD group without pretreatment,had no difference with both RIN and 28s/18s Ratio as indicator.The 100% ethanol pretreatment group was still the highest when using 28s/18s Ratio as indicator,but which had no statistic meaning.Conclusion： Our experiment preliminary proven that 100% ethanol processing method was more suitable for LMD cutting sample pretreatment compared with other two groups,and it could significantly reduce tissue RNA integrity lost during the cutting process.This step will helpful to maintain the quality of the samples for higher requirement of subsequent high throughput experimental.