Petersen Patrick Hannibal Dalsbo, James Jaslin Pallikkunnath, Riis Lene Buhl, Høgdall Claus Kim, Høgdall Estrid Vilma
Department of Pathology, Herlev University Hospital, 2730 Herlev, Denmark.
Department of Gynecology, The Juliane Marie Centre, Rigshospitalet, University of Copenhagen, 2100 Copenhagen, Denmark.
Methods Protoc. 2024 Dec 19;7(6):101. doi: 10.3390/mps7060101.
High-quality RNA is crucial in clinical diagnostics and precision medicine. Formalin-fixed and paraffin-embedded (FFPE) tissues pose a challenge due to nucleic acid fragmentation and crosslinking. In this pilot study, various commercially available techniques for extracting RNA from small FFPE samples were compared. We evaluated the KingFisher Duo automated system or the manual MagMAX FFPE DNA/RNA Ultra Kit as an RNA extraction method combined with either a xylene, d-limonene, or AutoLys M tubes deparaffinization method. Additionally, the automated Maxwell RSC RNA FFPE kit and the High Pure FFPET RNA Isolation Kit were examined using FFPE samples from inflammatory bowel disease (IBD) patients, as well as samples from ovarian, kidney, and breast cancer and the skin. The KingFisher Duo system gave a higher yield and more consistent RNA quantities, especially from small volumes of IBD samples, compared to manual extraction. The deparaffinization method also impacted results, with AutoLys M tubes proving effective in combination with the KingFisher Duo system. Conversely, the High Pure kit exhibited higher yields for larger FFPE samples. While RNA integrity is a critical factor, particularly for messenger RNA (mRNA) expression studies, its role is less prominent in microRNA (miRNA) analyses. Recognizing this, our study focused on RNA yield and purity (A260/A230) to evaluate RNA extraction methods for various sample types. These findings emphasize the importance of selecting appropriate RNA extraction methods based on sample characteristics and research goals, highlighting the performance of automated methods and the impact of deparaffinization choices. The findings contribute to refining RNA extraction for molecular biology analyses, suggesting avenues for further exploration, including cost-effectiveness under specific experimental conditions.
高质量RNA在临床诊断和精准医学中至关重要。由于核酸片段化和交联，福尔马林固定石蜡包埋（FFPE）组织带来了挑战。在这项初步研究中，比较了从少量FFPE样本中提取RNA的各种市售技术。我们评估了KingFisher Duo自动化系统或手动MagMAX FFPE DNA/RNA Ultra试剂盒作为RNA提取方法，并结合二甲苯、d-柠檬烯或AutoLys M管脱石蜡方法。此外，使用来自炎症性肠病（IBD）患者的FFPE样本以及卵巢癌、肾癌、乳腺癌和皮肤样本，对自动化Maxwell RSC RNA FFPE试剂盒和高纯FFPET RNA分离试剂盒进行了检测。与手动提取相比，KingFisher Duo系统产量更高，RNA量更一致，尤其是从小体积的IBD样本中提取时。脱石蜡方法也会影响结果，事实证明AutoLys M管与KingFisher Duo系统结合使用有效。相反，高纯试剂盒对较大的FFPE样本产量更高。虽然RNA完整性是一个关键因素，特别是对于信使RNA（mRNA）表达研究，但它在微小RNA（miRNA）分析中的作用不太突出。认识到这一点，我们的研究侧重于RNA产量和纯度（A260/A230），以评估针对各种样本类型的RNA提取方法。这些发现强调了根据样本特征和研究目标选择合适的RNA提取方法的重要性，突出了自动化方法的性能以及脱石蜡选择的影响。这些发现有助于改进用于分子生物学分析的RNA提取，为进一步探索提供了途径，包括特定实验条件下的成本效益。
