随着分子生物学相关检测技术的发展,以miRNA为主的非编码RNA(ncRNA)可为组织体液鉴定[1-4]、死亡时间相关推断[5-7]、年龄估计[8-10]等提供新的策略和方法,在法医学研究中发挥重要作用。但低质量的RNA会增加基因表达分析的难度,影响研究结果[11-13]。因此RNA的完整性是成功分析转录组和获得RNA标记物准确表达数据的先决条件。 Science International发表了题为“RNA quality score evaluation: A preliminary study of RNA integrity number (RIN) and RNA integrity and quality number (RNA IQ)”的研究论文,该研究评估了RIN值和RNA IQ值在RNA样本完整性检测中的性能,并通过迈克生物D 600全自动数字PCR分析系统评估了不同降解程度对RNA样本中3个miRNA表达水平的影响,为RNA生物标志物的分析提供了可靠的检测方法[14]。 同时,研究者利用qPCR和迈克生物D 600全自动数字PCR分析系统(图a)检测了完整和严重降解RNA样本中三种miRNA(hsa-let-7g-5p、hsa-miR-93-5p和hsa-miR-191-5p)的表达水平。结果显示,与完整的RNA样本相比,严重降解的样本中三种miRNA的表达水平没有明显变化(图b),因此分析具有稳定区域或微小的RNA生物标志物对于降解程度较高的样本(例如体液斑等)是可靠的研究方式。 基于标准曲线法和依赖Ct值的间接定量方法,以及PCR抑制剂的存在使得qPCR过程繁琐且容易导至结果不稳定。而数字PCR作为第三代PCR技术,克服了qPCR的局限性,并且灵敏度更高,可直接获取目标基因的绝对拷贝数,为miRNA研究提供了更高的检测精度及分辨率,全自动的检测设备也极大降低了操作的复杂性,成为评估miRNA完整性的极具应用价值的检测新方法。 迈克生物D 600全自动数字PCR分析系统采用自主研发的振动微滴(OsciDrop®)核心专利技术,实现了核酸的绝对定量、灵敏度高、耐受抑制物干扰,并且集成了微滴生成、PCR扩增和荧光检测三大步骤,实现了“样本进、结果出”的全新模式。目前迈克生物数字PCR平台已开展30余项科研试剂产品研发,涉及肿瘤、遗传性疾病、病原体检测等疾病领域,并可提供个性化试剂定制服务,能够满足临床、科研、疾控、司法鉴定及食品安全等多应用场景的精准检测需求。 Multiplex mRNA profiling for the identification of body fluids, Forensic Sci. Int. 152 (2005) 1–12. Maake,et al.mRNA profiling for body fluid identification by reverse transcription endpoint PCR and realtime PCR, Forensic Sci. Int. Genet. 3 (2009) 80-88. The development of a mRNA multiplex RT-PCR assay for the definitive identification of body fluids, Forensic Sci. Int. Genet. 4 (2010) 244-256. Ballantyne, Identification of forensically relevant body fluids using a panel of differentially expressed microRNAs, Anal. Biochem. 387 (2009) 303-314. Teti,et al.HIF1α protein and mRNA expression as a new marker for post mortem interval estimation in human gingival tissue, J. Anat. 232 (2018) 1031-1037. al.Evaluating the potential of housekeeping genes, rRNAs, snRNAs, microRNAs and circRNAs as reference genes for the estimation of PMI, Forensic Sci. Med. Pathol. 14 (2018) 194-201. Vison`a,et al.The thanatotranscriptome: gene expression of male reproductive organs after death, Gene 675 (2018) 191-196. S. D,et al. Extracellular RNA profiles with human age, Aging Cell 17 (2018) e12785. Chatsirisupachai, D. Palmer,et al. Ageing-associated changes in the expression of lncRNAs in human tissues reflect a transcriptional modulation in ageing pathways, Mech. Ageing Dev. 185 (2020) 111177. Huan, G. Chen, C. Liu,et al.Ageassociated microRNA expression in human peripheral blood is associated with allcause mortality and age-related traits, Aging Cell 17 (2018) e12687. Romero, A.A. Pai, J. Tung,et al.RNA-seq: impact of RNA degradation on transcript quantification, BMC Biol. 12 (2014) 42. Fleige, V. Walf, S. Huch,,et al.Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR, Biotechnol. Lett. 28 (2006) 1601-1613. Chen, T. Souaiaia, J.S. Herstein, et al., Effect of RNA integrity on uniquely mapped reads in RNA-Seq, BMC Res. Notes 7 (2014) 753. Li, J Liu,M-Y Zhao,et al.RNA quality score evaluation: A preliminary study of RNA integrity number (RIN) and RNA integrity and quality number (RNA IQ),Forensic Science International,357 (2024) 111976 |