从福尔马林固定石蜡包埋的乳腺癌组织中双重提取 DNA 和 RNA 的结果和经验教训，用于一项大型癌症流行病学研究。
Results and lessons from dual extraction of DNA and RNA from formalin-fixed paraffin-embedded breast tumor tissues for a large Cancer epidemiologic study.
机构信息
Department of Cancer Prevention and Control, Roswell Park Comprehensive Cancer Center, Elm & Carlton Streets, Buffalo, NY, 14263, USA.
Center for Inherited Disease Research, Johns Hopkins Genomics, Institute of Genetic Medicine, Johns Hopkins School of Medicine, Baltimore, MD, USA.
出版信息
BMC Genomics. 2022 Aug 25;23(1):614. doi: 10.1186/s12864-022-08837-6.
BACKGROUND
The use of archived formalin-fixed paraffin-embedded (FFPE) tumor tissues has become a common practice in clinical and epidemiologic genetic research. Simultaneous extraction of DNA and RNA from FFPE tissues is appealing but can be practically challenging. Here we report our results and lessons learned from processing FFPE breast tumor tissues for a large epidemiologic study.
METHODS
Qiagen AllPrep DNA/RNA FFPE kit was adapted for dual extraction using tissue punches or sections from breast tumor tissues. The yield was quantified using Qubit and fragmentation analysis by Agilent Bioanalyzer. A subset of the DNA samples were used for genome-wide DNA methylation assays and RNA samples for sequencing. The QC metrices and performance of the assays were analyzed with pre-analytical variables.
RESULTS
A total of 1859 FFPE breast tumor tissues were processed. We found it critical to adjust proteinase K digestion time based on tissue volume to achieve balanced yields of DNA and RNA. Tissue punches taken from tumor-enriched regions provided the most reliable output. A median of 1475 ng DNA and 1786 ng RNA per sample was generated. The median DNA integrity number (DIN) was 3.8 and median DV200 for RNA was 33.2. Of 1294 DNA samples used in DNA methylation assays, 97% passed quality check by qPCR and 92% generated data deemed high quality. Of the 130 RNA samples with DV200 ≥ 20% used in RNA-sequencing, all but 5 generated usable transcriptomic data with a mapping rate ≥ 60%.
CONCLUSIONS
Dual DNA/RNA purification using Qiagen AllPrep FFPE extraction protocol is feasible for clinical and epidemiologic studies. We recommend tissue punches as a reliable source material and fine tuning of proteinase K digestion time based on tissue volume.
IMPACT
Our protocol and recommendations may be adapted by future studies for successful extraction of archived tumor tissues.
背景
在临床和流行病学遗传研究中，使用存档的福尔马林固定石蜡包埋（FFPE）肿瘤组织已成为一种常见做法。从 FFPE 组织中同时提取 DNA 和 RNA 很有吸引力，但在实践中可能具有挑战性。在这里，我们报告了从大量流行病学研究的 FFPE 乳腺肿瘤组织中进行处理的结果和经验教训。
方法
Qiagen AllPrep DNA/RNA FFPE 试剂盒经过改良，可使用组织打孔器或乳腺肿瘤组织切片进行双重提取。使用 Qubit 定量提取产物，并通过 Agilent Bioanalyzer 进行片段分析。使用一部分 DNA 样本进行全基因组 DNA 甲基化分析，使用 RNA 样本进行测序。使用预分析变量分析 QC 度量标准和测定性能。
结果
共处理了 1859 例 FFPE 乳腺肿瘤组织。我们发现，根据组织体积调整蛋白酶 K 消化时间以实现 DNA 和 RNA 的平衡产量非常关键。从富含肿瘤的区域采集的组织打孔器提供了最可靠的输出。每个样本平均产生 1475ng DNA 和 1786ng RNA。中位数 DNA 完整性数（DIN）为 3.8，中位数 RNA 的 DV200 为 33.2。在用于 DNA 甲基化分析的 1294 个 DNA 样本中，97%通过 qPCR 质量检查，92%生成被认为是高质量的数据。在用于 RNA-seq 的 130 个 RNA 样本中，除 5 个外，所有 RNA 的 DV200≥20%，并且均生成了具有≥60%映射率的可用转录组数据。
结论
使用 Qiagen AllPrep FFPE 提取方案进行双重 DNA/RNA 纯化对于临床和流行病学研究是可行的。我们建议使用组织打孔器作为可靠的源材料，并根据组织体积调整蛋白酶 K 消化时间。
影响
我们的方案和建议可能被未来的研究采用，以成功提取存档的肿瘤组织。
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