Institute of Forensic Medicine, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu 610041, China.
Institute of Forensic Medicine, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu 610041, China; College of Forensic Medicine, Hebei Medical University, Hebei Key Laboratory of Forensic Medicine, Shijiazhuang 050017, China.
Forensic Sci Int. 2024 Apr;357:111976. doi: 10.1016/j.forsciint.2024.111976. Epub 2024 Mar 2.
In the past several years, with the in-depth development of RNA-related research, exploring the application of transcriptome and corresponding RNA biomarkers has become one of the research hotspots in the field of forensic science. High-quality RNA is essential for successful downstream workflows, especially in the steps of screening biomarkers by microarray or RNA sequencing (RNA-seq). Thus, accurately evaluating the quality of RNA samples is a critical step in obtaining meaningful expression data. The RNA integrity number (RIN) generated from the Agilent Bioanalyzer system has been widely used for RNA quality control in the past two decades. Recently, Thermo Fisher Scientific launched a ratiometric fluorescence-based method to quickly check whether an RNA sample has degraded, and the results are presented as RNA integrity and quality number (RNA IQ). Both quality score systems determine RNA quality using a numerical system based on a scale of 1-10, with 1 denoting significantly degraded specimens and 10 representing high-quality, intact RNA samples. In this preliminary study, we evaluated the consistency, reproducibility and linearity of two quality scores in RNA quality determination by analyzing heat- and RNase- artificially degraded samples. Meanwhile, the expression levels of three microRNAs (hsa-let-7 g-5p, hsa-miR-93-5p and hsa-miR-191-5p) in intact and severely degraded RNA samples were estimated by TaqMan-qPCR and droplet digital PCR. Overall, both quality scores showed good repeatability and reproducibility in their respective tests. In the samples subjected to thermal degradation, RIN showed a trend corresponding to heating time, while RNA IQ value showed almost no change on the time gradient. However, in RNase A mediated degradation, RNA IQ value observed better linearity. Furthermore, the expression levels of three microRNAs in the severely degraded samples did not show significant changes compared to the intact RNA samples. RNA degradation is a very complex and highly variable process, which is difficult to comprehensively evaluate through any one index and cannot directly compare these two parameters. Nevertheless, combined with previous research results and the expression levels of three microRNAs in this study, analyzing RNA biomarkers with stable regions or small sizes in challenged samples may be a conservative and reliable approach.
在过去的几年中，随着 RNA 相关研究的深入，探索转录组和相应 RNA 生物标志物的应用已成为法医学领域的研究热点之一。高质量的 RNA 对于下游工作流程的成功至关重要，特别是在通过微阵列或 RNA 测序（RNA-seq）筛选生物标志物的步骤中。因此，准确评估 RNA 样本的质量是获得有意义的表达数据的关键步骤。在过去的二十年中，Agilent Bioanalyzer 系统生成的 RNA 完整性编号（RIN）已被广泛用于 RNA 质量控制。最近，赛默飞世尔科技推出了一种基于比率荧光的快速检查方法，用于检查 RNA 样本是否降解，结果以 RNA 完整性和质量编号（RNA IQ）表示。这两种质量评分系统均使用基于 1-10 刻度的数值系统来确定 RNA 质量，其中 1 表示严重降解的标本，10 表示高质量、完整的 RNA 样本。在这项初步研究中，我们通过分析热和 RNase 人为降解的样本，评估了两种质量评分在 RNA 质量测定中的一致性、可重复性和线性。同时，通过 TaqMan-qPCR 和数字液滴 PCR 估计了完整和严重降解 RNA 样本中三种 microRNA（hsa-let-7g-5p、hsa-miR-93-5p 和 hsa-miR-191-5p）的表达水平。总体而言，两种质量评分在各自的测试中均表现出良好的可重复性和重现性。在受热降解的样品中，RIN 显示出与加热时间相对应的趋势，而 RNA IQ 值在时间梯度上几乎没有变化。然而，在 RNase A 介导的降解中，RNA IQ 值表现出更好的线性。此外，与完整 RNA 样本相比，严重降解样本中三种 microRNA 的表达水平没有明显变化。RNA 降解是一个非常复杂且高度可变的过程，很难通过任何一个指标进行全面评估，也不能直接比较这两个参数。然而，结合之前的研究结果和本研究中三种 microRNA 的表达水平，在受挑战的样本中分析具有稳定区域或较小大小的 RNA 生物标志物可能是一种保守且可靠的方法。
