摘要
目的评估外周血不同预处理方法以及储存时长对RNA质量的影响,优化外周血的保存方法。方法选取8种预处理方法对3名健康无关个体的外周血进行预处理并保存在-80℃条件下,使用Quick-RNA^(TM) Miniprep Plus试剂盒提取样本总RNA。采用外周血添加DNA/RNA Shield^(TM)的方法对外周血样本进行处理后于-80℃条件下分别保存0、5、10、15、30和60 d后抽提总RNA并测定RNA的浓度、纯度以及完整性。采用SPSS 22.0软件对RNA的产量、纯度以及完整性进行比较。结果纯度方面,白细胞加RNAlater^(TM)法和直接冻存法最差,其余6种方法纯度较好;产量方面,血细胞加DNA/RNA Shield^(TM)法产量最高,外周血加DNA/RNA Shield^(TM)法次之;完整性方面,PAXgene全血RNA管法最好,除外周血加DNA/RNA Shield^(TM)法和血细胞加DNA/RNA Shield^(TM)法外,其余5种与PAXgene全血RNA管法比较差异均具有统计学意义。6个储存时长的RNA纯度均在1.815~1.952,随着储存时长的增加,RNA产量和完整性均下降,其中产量从4.516ng降至1.039ng,完整性由8.533降至7.150。结论综合RNA产量、纯度及完整性等因素,加DNA/RNA Shield^(TM)是一种较为理想的外周血预处理方法。在此基础上,样本可在低温条件下储存最多60 d。
Objective To evaluate the effects of different pretreatment methods and preservation time on RNA quality of peripheral blood samples,and to optimize the preservation method of peripheral blood samples.Methods Eight pretreatment methods were used to preprocess the peripheral blood from 3 healthy unrelated individuals and the treated samples were stored at-80℃.Total RNA of samples was extracted using Quick-RNA^(TM) Miniprep Plus kit.DNA/RNA Shield^(TM) was added to peripheral blood and total RNA was extracted after preservation at-80℃for 0,5,10,15,30 and 60 days,respectively.The concentration,purity and integrity of RNA were determined.Statistical analyses were performed by SPSS 22.0 software to compare the differences in RNA yield,purity and integrity among the eight pretreatment methods.Results In terms of purity,leukocyte pretreated with RNAlater^(TM) and directly cryopreservation peripheral blood showed the worst purity.The other six methods showed better purity.In terms of yield,blood cells with DNA/RNA Shield^(TM) came out with the highest yield,followed by peripheral blood with DNA/RNA Shield^(TM).In terms of integrity,peripheral blood preserved in PAXgene Blood RNA tube method had the best integrity.Except for peripheral blood pretreated with DNA/RNA Shield^(TM) and blood cells pretreated with DNA/RNA shield^(TM),the other five methods had statistical differences when compared to the method by keeping peripheral blood in PAXgene Blood RNA tube.The purity of RNA stored at six-time gradients ranged from 1.815 to 1.952.With the increase of storage time,RNA yield decreased from 4.516 ng to 1.039 ng,and RNA integrity decreased from 8.533 to 7.150.Conclusion According to the results of total RNA’s yield,purity and integrity,peripheral blood pretreated with DNA/RNA Shield^(TM) was the best pretreatment method.After the pretreatment,samples can be preserved for up to 60 days in low temperature.
作者
张佳怡
徐倩南
刘希玲
李成涛
ZHANG Jia-yi;XU Qian-nan;LIU Xi-ling;LI Cheng-tao(Shanghai Key Laboratory of Forensic Medicine,Key Laboratory of Forensic Science,Ministry of Justice,Shanghai Forensic Service Platform,Academy of Forensic Science,Shanghai 200063,China;Department of Forensic Medicine,Inner Mongolia Medical University,Hohhot 010030,China;School of Forensic Medi-cine,Southern Medical University,Guangzhou 510515,China;West China School of Basic Medical Sciences&Forensic Medicine,Sichuan University,Chengdu 610041,China)
出处
《法医学杂志》
CAS
CSCD
2021年第6期825-831,共7页
Journal of Forensic Medicine
基金
国家自然科学基金资助项目(81871534)
中央级科研院所公益专项(GY2021G-2)
司法部司法鉴定重点实验室资助项目
上海市法医学重点实验室资助项目(21DZ2270800)
上海市司法鉴定专业技术服务平台资助项目(19DZ2292700)。
关键词
法医遗传学
核糖核酸
预处理
储存时长
外周血
forensic genetics
ribonucleic acid(RNA)
pretreatment
storage time
peripheral blood
