目的 比较7种不同的RNA提取方法（6种商业化试剂盒加Trizol有机提取法）的相对提取效率,为法医实践中选择提取高质量的RNA/miRNA提供参考。方法 分别使用6种不同的商业化试剂盒以及Trizol有机提取法提取晾干的外周血棉签样本,通过对RNA进行紫外荧光定量检测、琼脂糖凝胶电泳检测和测定样本中内参基因RNU6b的相对含量等方式进行评价。结果 各试剂盒所提RNA/miRNA的质量评估和表达分析结果有明显差异,其中Trizol有机提取法和试剂盒RNeasy®Mini可以得到更高质量和纯度的RNA/miRNA,170µL外周血中所提取的RNA总量可达到（3860.50±182.12）ng、纯度(OD260/280)约为1.84±0.03,且琼脂糖电泳结果显示RNA完整性好,在后续实验中表现良好。结论 在所有的提取方法中,综合考虑RNA的数量、质量以及Realtime PCR检测结果,更适合提取外周血RNA的方法为RNeasy® Mini Kit和Trizol有机提取法。
Abstract
Objective To compare the seven commonly-used commercially-available RNA extraction kits and relevant methods (six commercially available extraction kits plus Trizol organic extraction suite) so as to assess their relative effectiveness of yielding RNA/miRNA. Methods Dried peripheral bloods were taken as the samples for the RNA to extract respectively by the seven selected methods. RNA quality was evaluated with UV spectrophotometry, agar-gel electrophoresis and quantitative PCR expression analysis. Results It was showed that different methods exhibit considerable discrepancy for either the quality or expression levels. Both Trizol organic RNA extraction suite and RNeasy® Mini Kit can get RNA/miRNA of good quantity and quality (From 170µL peripheral blood, the total obtained RNA quantity >1353.50 ± 78.87ng, OD260/280 >1.84 ± 0.03), rendering the yielded RNA to be of good integrity as revealed by agar-gel electrophoresis. Conclusion From overall comprehension by the quantity, quality and quantitative PCR-based expression analysis, the RNeasy® Mini Kit and Trizol are more suitable for extracting RNA/miRNA from peripheral blood.
关键词
法医遗传学 /
微RNA /
提取方法 /
定量PCR技术
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Key words
forensic genetics /
microRNA /
extraction method /
quantitative PCR
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参考文献
[1] HE L, HANNON G J.MiRNAs: small RNAs with a big role in gene regulation[J]. Nature Reviews Genetics. 2004, 5: 522-531.
[2] ZUBAKOV D, KOKSHOORN M, KLOOSTERMAN A, et al.New markers for old stains: stable mRNA markers for blood and saliva identification from up to 16-year-old stains[J]. International Journal of Legal Medicine, 2009, 123: 71-74.
[3] 陈琦,易少华,黄代新. microRNAs及其法医学应用前景[J]. 中国法医学杂志, 2015, 27(3): 215-218.
[4] 高林林,李佑英,严江伟,等. RNA在法医学领域中的应用及研究进展[J]. 法医学杂志, 2011, 27(6): 455-459.
[5] HANSON E K, LUBENOW H, BALLANTYNE J.Identification of forensically relevant body fluids using a panel of differentially expressed microRNAs[J]. Analytical Biochemistry, 2009, 387: 303-314.
[6] WANG Z, ZHANG J, LUO H, et al.Screening and confirmation of microRNA markers for forensic body fluid identification[J]. Forensic Science International Genetics, 2013, 7:116-123.
[7] 肖旭峰,姜文轩,吴才君,等. 野葛块根总RNA的不同提取方法比较研究[J]. 江西农业大学学报, 2011, 33(1): 147-150.
[8] 何松哲,何慧,陈懿,等. 不同保存和处理方式对全血标本总RNA提取的影响[J]. 中国微生态学杂志, 2015, 27(2): 223-226.
[9] JIANG Z, UBOH C, CHEN J, et al.Isolation of RNA from equine peripheral blood cells: comparison of methods[J]. Springer Plus, 2013, 478.
[10] 白雪,张丽颖,刘莉娜,等. 组织中RNA提取过程中影响因素的研究[J]. 黑龙江医药科学, 2015, 38(4): 12-13.
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脚注
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基金
“十三五”国家重点研发计划项目（No. 2017YFC0803503）; 公安部科技强警项目（No. 2016GABJC17）; 中央级公益性科研院所基本科研业务费专项资金项目（No. 2016JB038,No. 2017JB003）
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