Screening and confirmation of microRNA markers for forensic body fluid identification.
Screening and confirmation of microRNA markers for forensic body fluid identification.
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DOI：
10.1016/j.fsigen.2012.07.006
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发表时间：
2013
期刊：
影响因子：
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通讯作者：
Z. Wang;Ji Zhang;Haibo Luo;Y. Ye;Jing Yan;Yiping Hou
中科院分区：
文献类型：
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作者：
Z. Wang;Ji Zhang;Haibo Luo;Y. Ye;Jing Yan;Yiping Hou
MicroRNAs(miRNAs，∼22核苷酸)是一种小的、非蛋白质编码的RNA，在转录后水平上调节基因的表达。MiRNAs可以以组织特异性的方式表达，并已被引入法医体液鉴定。在本研究中，我们使用qPCR阵列(TaqMan®阵列人microRNA卡)来筛选体液特异的miRNAs。7个候选miRNAs被鉴定为潜在的体液特异性，可用作法医学相关的体液标志物：静脉血的miR16和miR486，精液的miR888和miR891a，月经血的miR214，阴道分泌物的miR124a，唾液的miR138-2。然后通过水解探针定量实时聚合酶链式反应(TaqMan-qPCR)对候选miRNA标记进行验证。此外，使用BestKeeper软件验证了四个基因RNU44、RNU48、U6和U6b的表达稳定性，这四个基因通常被用作法医体液研究的参考基因(RGS)。本研究表明，U6可作为法医体液鉴定中miRNAs的合适RG。MiR486、miR888、miR214、miR16和miR891a的相对表达比(R)可以将目标体液与本研究中检测的其他体液区分开来。在5个已确认的miRNA标记中，TaqMan-qPCR的检测限为总RNA的10pg。在正常实验室条件下，对血痕和精斑进行1个月的时间降解效果测试，对检测结果无明显影响。在此，本研究提出了5种体液特异性miRNAs用于静脉血、精液和月经血的法医学鉴定，其中miR486、miR888和miR214可作为体液鉴定的新标记物。在寻找合适的miRNA标记和稳定的RGS用于法医体液鉴定方面仍有必要进行额外的工作。
MicroRNAs (miRNAs, ∼22 nucleotides) are small, non-protein coding RNAs that regulate gene expression at the post-transcriptional level. MiRNAs can express in a tissue-specific manner, and have been introduced to forensic body fluid identification. In this study, we employed the qPCR-array (TaqMan®Array Human MicroRNA Cards) to screen the body fluid-specific miRNAs. Seven candidate miRNAs were identified as potentially body fluid-specific and could be used as forensically relevant body fluid markers: miR16 and miR486 for venous blood, miR888 and miR891a for semen, miR214 for menstrual blood, miR124a for vaginal secretions, and miR138-2 for saliva. The candidate miRNA markers were then validated via hydrolysis probes quantitative real-time polymerase chain reaction (TaqMan-qPCR). In addition, BestKeeper software was used to validate the expression stability of four genes, RNU44, RNU48, U6 and U6b, regularly used as reference genes (RGs) for studies involving forensic body fluids. The current study suggests that U6 could be used as a proper RG of miRNAs in forensic body fluid identification. The relative expression ratios (R) of miR486, miR888, miR214, miR16 and miR891a can differentiate the target body fluid from other body fluids that were tested in this study. The detection limit of TaqMan-qPCR of the five confirmed miRNA markers was 10pg of total RNA. The effect of time-wise degradation of blood stains and semen stains for 1 month under normal laboratory conditions was tested and did not significantly affect the detection results. Herein, this study proposes five body fluid-specific miRNAs for the forensic identification of venous blood, semen, and menstrual blood, of which miR486, miR888, and miR214 may be used as new markers for body fluid identification. Additional work remains necessary in search for suitable miRNA markers and stable RGs for forensic body fluid identification.