Decruyenaere Philippe, Verniers Kimberly, Poma-Soto Franco, Van Dorpe Jo, Offner Fritz, Vandesompele Jo
Department of Hematology, Ghent University Hospital, Ghent, Belgium; OncoRNALab, Cancer Research Institute Ghent, Ghent University, Ghent, Belgium; Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.
OncoRNALab, Cancer Research Institute Ghent, Ghent University, Ghent, Belgium; Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.
Lab Invest. 2023 Feb;103(2):100027. doi: 10.1016/j.labinv.2022.100027. Epub 2023 Jan 10.
Archived formalin-fixed, paraffin-embedded (FFPE) tissue samples are being increasingly used in molecular cancer research. Compared with fresh-frozen tissue, the nucleic acid analysis of FFPE tissue is technically more challenging. This study aimed to compare the impact of 3 different RNA extraction methods on yield, quality, and sequencing-based gene expression results in FFPE samples. RNA extraction was performed in 16 FFPE tumor specimens from patients with diffuse large B-cell lymphoma and in reference FFPE material from microsatellite-stable and microsatellite-instable cell lines (3 replicates each) using 2 silica-based procedures (A, miRNeasy FFPE; C, iCatcher FFPE Tissue RNA) and 1 isotachophoresis-based procedure (B, Ionic FFPE to Pure RNA). The RNA yield; RNA integrity, as reflected by the distribution value 200; and RNA purity, as reflected by the 260/280 and the 260/230 nm absorbance ratios, were determined. The RNA was sequenced on the NovaSeq 6000 instrument using the TruSeq RNA Exome and SMARTer Stranded Total RNA-Seq Pico v3 library preparations kits. Our results highlight the impact of RNA extraction methodology on both preanalytical and sequencing-based gene expression results. Overall, methods B and C outperformed method A because these showed significantly higher fractions of uniquely mapped reads, an increased number of detectable genes, a lower fraction of duplicated reads, and better representation of the B-cell receptor repertoire. Differences among the extraction methods were generally more explicit for the total RNA sequencing method than for the exome-capture sequencing method. Importantly, the predicative value of quality metrics varies among extraction kits, and caution should be applied when comparing and interpreting results obtained using different methods.
存档的福尔马林固定石蜡包埋（FFPE）组织样本在分子癌症研究中的应用越来越广泛。与新鲜冷冻组织相比，FFPE组织的核酸分析在技术上更具挑战性。本研究旨在比较3种不同RNA提取方法对FFPE样本的产量、质量以及基于测序的基因表达结果的影响。使用2种基于硅胶的方法（A，miRNeasy FFPE；C，iCatcher FFPE Tissue RNA）和1种基于等速电泳的方法（B，Ionic FFPE to Pure RNA），对16例弥漫性大B细胞淋巴瘤患者的FFPE肿瘤标本以及微卫星稳定和微卫星不稳定细胞系的对照FFPE材料（各3份重复样本）进行RNA提取。测定RNA产量；用分布值200反映的RNA完整性；以及用260/280和260/230 nm吸光度比值反映的RNA纯度。使用TruSeq RNA Exome和SMARTer Stranded Total RNA-Seq Pico v3文库制备试剂盒在NovaSeq 6000仪器上对RNA进行测序。我们 的结果突出了RNA提取方法对分析前和基于测序的基因表达结果的影响。总体而言，方法B和C优于方法A，因为它们显示出独特映射 reads 的比例显著更高、可检测基因数量增加、重复 reads 的比例更低以及B细胞受体库的代表性更好。提取方法之间的差异在总RNA测序方法中通常比在外显子捕获测序方法中更明显。重要的是，质量指标的预测价值在不同提取试剂盒之间有所不同，在比较和解释使用不同方法获得的结果时应谨慎。
