Comparison of automated and manual purification of total RNA for mRNA-based identification of body fluids
Comparison of automated and manual purification of total RNA for mRNA-based identification of body fluids
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DOI：
10.1016/j.fsigen.2014.09.007
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发表时间：
2015-01-01
影响因子：
3.1
通讯作者：
Sakurada, Koichi
中科院分区：
文献类型：
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作者：
Akutsu, Tomoko;Kitayama, Tetsushi;Sakurada, Koichi
基于二氧化硅柱的RNA纯化程序已广泛应用于法医样本体液鉴定的mRNA分析。此外，采用磁珠技术的自动化RNA纯化系统最近也变得可用。在本初步研究中，为了确定哪种RNA纯化技术更适用于实时逆转录聚合酶链反应（RT-PCR）对体液的鉴定，我们对使用EZ1 Advanced Instrument自动纯化和使用RNeasy Mini Kit手动纯化的总RNA的产量和质量进行了比较分析。通过对两种不同大小的β -肌动蛋白基因片段的基因表达分析，比较了总RNA的产量和大小分布。此外，比较了几种纯化方法中几个目标基因的相对量，并通过芯片电泳测定了总RNA的完整性。本研究结果表明，与使用EZ1的自动纯化相比，RNeasy可以纯化更高质量的RNA。然而，在EZ1纯化的样品中，RT-PCR分析的灵敏度更高，这可能是由于EZ1从降解样品中提取短长度RNA的相对效率。我们还表明，体液特异性基因的相对水平的定量可能受到纯化程序的影响。我们的研究结果表明，尽管在基因表达分析中通常需要使用高质量的RNA来获得可重复的结果，但不能排除高度降解样品中短RNA片段的法医相关性。此外，我们的研究结果表明，自动纯化程序以及基于二氧化硅柱的人工纯化程序可用于法医样本中基于mrna的体液鉴定。2014爱思唯尔爱尔兰有限公司版权所有。
Silica column-based RNA purification procedures have widespread use in mRNA profiling for body fluid identification in forensic samples. Also, automated RNA purification systems employing magnetic bead technology have recently become available. In this preliminary study, to ascertain which RNA purification technology is more suitable for the identification of body fluids by real-time reverse transcription polymerase chain reaction (RT-PCR), comparative analyses of the yield and quality of total RNA were performed between automated purification using an EZ1 Advanced Instrument and manual purification using an RNeasy Mini Kit. The yield and size distribution of total RNA were compared by gene expression analysis of two different sized fragments of the beta-actin gene. In addition, the relative amounts of several target genes were compared between the purification methods, and the integrity of total RNA was determined by chip-based electrophoresis. The results of this study suggest that RNeasy can purify higher-quality RNA as compared with automated purification using EZ1. The sensitivity of the RT-PCR analysis, however, was higher in the EZ1-purified samples, likely due to the relative efficiency of EZ1 in extracting short-length RNA from degraded samples. We also show that the quantification of relative levels of body fluid-specific genes could be influenced by the purification procedure. Our results indicate that although use of high-quality RNA is generally required for reproducible results in gene expression analysis, the forensic relevance of short RNA fragments in highly degraded samples cannot be ruled out. Furthermore, our results suggest that automated purification procedures as well as silica column-based manual purification procedures can be used for mRNA-based body fluid identification in forensic samples. (C) 2014 Elsevier Ireland Ltd. All rights reserved.