摘要
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 investigate if different RNA extraction methods could affect the total RNA concentration and integrity of samples dissected by LMD system from murine tooth germs.Methods According to the different RNA extraction methods,the samples were divided into three groups： RNA extraction kit group,classic Trizol method group and modified Trizol method group.The tooth germ tissues of the mandibular first molars of CD1 mice at E17 and PN2 stages were dissected by LMD6000 system.Total RNA was extracted from each group in the 3 ways.To ensure the integrity of RNA samples,quality check was adopted with Bioanalyzer 2100 System,Agilent.The 28 S /18 S ratios of ribosome RNA,RNA integrity numbers（RIN） and the peak patterns of the electrophoretograms were integrated as the indices of RNA integrity.The RNA concentration and integrity of all the groups were compared with SPSS statistical analysis software.Results The RNA concentration in modified Trizol method group was the highest,which had significant difference compared with the other two groups.The classic Trizol method group had higher concentration than RNA extraction kit group,however,the difference didn＇t have statistical meaning.The comparison of RNA integrity among three groups showed no significant differences taking RIN numbers and 28 S /18 S ratios and electrophoretograms as indicators.Conclusions Our experiment preliminaryly proves that modified Trizol method can extract most total RNA compared with other two groups,while there is no stastically significant difference in RNA integrity among 3 methods.Modified Trizol method will be helpful to get chip experimental samples that meet the inspection standards for high flux experiment.
出处
《口腔医学》
CAS
2013年第7期446-449,共4页
Stomatology
基金
全军十一五课题重点项目(06MA004)
