Evaluation of a co-extraction kit for mRNA, miRNA and DNA methylation-based body fluid identification
Evaluation of a co-extraction kit for mRNA, miRNA and DNA methylation-based body fluid identification
复制标题
DOI：
10.1016/j.legalmed.2019.101630
复制
发表时间：
2020-02-01
期刊：
影响因子：
1.5
通讯作者：
Akutsu, Tomoko
中科院分区：
文献类型：
--
作者：
Watanabe, Ken;Akutsu, Tomoko
最近，信使RNA（mRNA）、微小RNA（miRNA）和DNA甲基化（DNAm）已被报道为体液鉴定（BFID）的新标记物。对这些标志物进行综合分析，可作为一种灵活、可靠的BFID方法，适用于各类法医学检材。然而，根据样本的可用量，独立提取所有靶标可能是困难的。在这项研究中，这些分子的共提取试剂盒，AllPrep DNA/RNA/miRNA通用试剂盒（APU）的适用性进行了评估，通过比较的RNA和DNA提取的血液和唾液污渍的APU与标准试剂盒提取的每种分子和先前报道的mRNA/DNA或miRNA/DNA共提取的方法。使用Bioanalyzer平台的电泳和实时PCR分析显示，APU在提取的RNA或DNA的质量以及mRNA、miRNA和DNA的提取效率方面与每个标准试剂盒几乎等同。此外，APU优于共提取方法，特别是在RNA完整性和miRNA提取效率方面。此外，焦磷酸测序显示，由APU提取的DNA的甲基化比率与由标准DNA提取试剂盒提取的DNA的甲基化比率没有不同。总体而言，APU适用于BFID分析中mRNA/miRNA/DNA标记物的全面分析。由于DNA指纹图谱也可用于DNA分型，APU在BFID中的灵活性和可靠性以及样品消耗效率方面可能是体液样品法医检查的最佳选择之一。
Recently, messenger RNA (mRNA), micro RNA (miRNA), and DNA methylation (DNAm) have been reported as novel markers for body fluid identification (BFID). Comprehensive analysis of these markers should be a flexible and reliable BFID method for various types of forensic samples. I lowever, independent extraction of all targets can be difficult depending on the usable amounts of samples. In this study, the applicability of a co-extraction kit for these molecules, the AllPrep DNA/RNA/miRNA Universal Kit (APU), was evaluated by comparing RNA and DNA extracted from blood and saliva stains by the APU with those extracted by standard kits for each molecule and by previously reported methods for mRNA/DNA or miRNA/DNA co-extraction. Electrophoresis using the Bioanalyzer platform and real-time PCR analysis revealed that the APU performed almost equivalently to each standard kit in the quality of RNA or DNA extracted and extraction efficiency of mRNAs, miRNAs, and DNA. Moreover, the APU outperformed the co-extraction methods, especially in RNA integrity and miRNA extraction efficiency. In addition, pyrosequencing revealed that the methylation ratios of DNA extracted by the APU were not different from those extracted by standard DNA extraction kits. Overall, the APU is applicable to comprehensive analysis of mRNA/miRNA/DNAm markers for BFID analysis. Because the DNA eluate can also be used for DNA typing, the APU may be among the best choices for forensic examination of body fluid samples in terms of its flexibility and reliability in BFID and efficiency in sample consumption.