利用 Xeno 内部阳性对照 (IPC) 对用于下游应用的非人灵长类 (NHP) 组织中的高通量 RNA 提取试剂盒进行比较分析。

IF 1.3 4区 医学 Q4 PHARMACOLOGY & PHARMACY
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引用次数: 0

摘要

核糖核酸(RNA)的提取和纯化在分子生物学、细胞和基因治疗中起着举足轻重的作用,RNA 的质量和完整性对下游应用至关重要。与人工方法相比,自动化高通量系统具有可扩展性和减少劳动力需求的潜力,因此备受关注。然而,在保持 RNA 产量和纯度的同时确保高通量能力、可重复性和可靠性仍然是一项挑战。本研究评估并比较了四种市售的基于磁珠的高通量 RNA 提取试剂盒在六种天真非人灵长类(NHP)组织基质(脑、心、肾、肝、肺和脾)中的性能。评估的重点是 RNA 纯度、产量和使用 Xeno 内部阳性对照(IPC)加标的提取效率(EE)。样品(约 50 毫克)经打珠匀浆后,在 KingFisher Flex 平台上按照制造商的方案进行处理,共进行 8 次重复。使用 NanoDrop® 分光光度计测量 RNA 纯度和产量,通过实时反转录定量聚合酶链反应(RT-qPCR)评估 EE。研究结果表明,所有测试提取试剂盒的 RNA 纯度都很高,但 RNA 产量却有很大差异。不同组织类型的提取效率存在差异,从脑到肺组织的提取效率呈下降趋势。这些结果凸显了精心选择试剂盒和优化方法对实现可靠的下游应用的重要性。MagMAX™ mirVana™ 总 RNA 分离试剂盒的准确性和可重复性最高,是要求高 RNA 质量和一致性的应用的首选。其他试剂盒,如 Maxwell® HT simplyRNA 试剂盒,在成本和性能之间取得了很好的平衡,但在精度方面有所折衷。这些发现强调了根据研究的具体需求选择合适的 RNA 分离方法的重要性,突出了准确的核酸提取在基因和细胞治疗研究中的关键作用。总之,本研究强调了影响 RNA 提取性能的关键因素,强调研究人员和从业人员在设计实验方案时需要同时考虑试剂盒性能和组织特征。这些见解有助于不断提高分子生物学和细胞/基因治疗应用中 RNA 提取方法的可重复性和可靠性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Comparative analysis of high-throughput RNA extraction kits in Naïve Non-Human Primate (NHP) tissues for downstream applications utilizing Xeno Internal Positive Control (IPC)

Ribonucleic acid (RNA) extraction and purification play pivotal roles in molecular biology and cell and gene therapy, where the quality and integrity of RNA are critical for downstream applications. Automated high-throughput systems have gained interest due to their potential for scalability and reduced labor requirements compared to manual methods. However, ensuring high-throughput capabilities, reproducibility, and reliability while maintaining RNA yield and purity remains challenging.

This study evaluated and compared the performance of four commercially available high-throughput magnetic bead-based RNA extraction kits across six types of naïve non-human primate (NHP) tissue matrices: brain, heart, kidney, liver, lung, and spleen. The assessment focused on RNA purity, yield, and extraction efficiency (EE) using Xeno Internal Positive Control (IPC) spiking.

Samples (∼50 mg) were homogenized via bead-beating and processed according to the manufacturer's protocol on the KingFisher Flex platform in eight replicates. RNA purity and yield were measured using a NanoDrop® spectrophotometer, while EE was evaluated via real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

The findings indicate consistent high RNA purity across all tested extraction kits, yet substantial variation in RNA yield. Extraction efficiency exhibited variations across tissue types, with decreasing trends observed from brain to lung tissues. These results underscore the importance of careful kit selection and method optimization for achieving reliable downstream applications. The MagMAX™ mirVana™ Total RNA Isolation Kit stands out as the most accurate and reproducible, making it the preferred choice for applications requiring high RNA quality and consistency. Other kits, such as the Maxwell® HT simplyRNA Kit, offer a good balance between cost and performance, though with some trade-offs in precision. These findings highlight the importance of selecting the appropriate RNA isolation method based on the specific needs of the research, underscoring the critical role of accurate nucleic acid extraction in gene and cell therapy research.

In conclusion, this study highlights the critical factors influencing RNA extraction performance, emphasizing the need for researchers and practitioners to consider both kit performance and tissue characteristics when designing experimental protocols. These insights contribute to the ongoing efforts to enhance the reproducibility and reliability of RNA extraction methods in molecular biology and cell/gene therapy applications.

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来源期刊
Journal of pharmacological and toxicological methods
Journal of pharmacological and toxicological methods PHARMACOLOGY & PHARMACY-TOXICOLOGY
CiteScore
3.60
自引率
10.50%
发文量
56
审稿时长
26 days
期刊介绍: Journal of Pharmacological and Toxicological Methods publishes original articles on current methods of investigation used in pharmacology and toxicology. Pharmacology and toxicology are defined in the broadest sense, referring to actions of drugs and chemicals on all living systems. With its international editorial board and noted contributors, Journal of Pharmacological and Toxicological Methods is the leading journal devoted exclusively to experimental procedures used by pharmacologists and toxicologists.
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