Messner Ella, Becker Lev, DeSanctis Mia L, Soranno Elizabeth A, Pianka Ryan, Pierce Caileigh, Johnson Molly, Poulin Rosalia Falco, Appiah-Madson Hannah J, Distel Daniel L
Ocean Genome Legacy Center, Marine and Environmental Sciences, Northeastern University, Nahant, Massachusetts, United States of America.
The Pennsylvania State University, University Park, Pennsylvania, United States of America.
PLoS One. 2025 Jun 3;20(6):e0321872. doi: 10.1371/journal.pone.0321872. eCollection 2025.
Cryopreservation is the gold standard for preserving high molecular weight (HMW) DNA (>10 kb) in tissue samples. However, frozen tissues are typically thawed either before or during DNA extraction, which can lead to substantial DNA degradation. In this study, we thawed the previously frozen tissues of 10 marine species (five fishes and five invertebrates) in the preservatives EDTA (250 mM, pH 10) or ethanol (EtOH, 95%) and maintained them in their respective preservatives overnight at 4°C before DNA extraction. We then compared the recovery of HMW DNA in these extracts to extracts prepared directly from frozen tissues. To evaluate the effect of these treatments on HMW DNA recovery, we determined the percentage of high molecular weight DNA (%HMW) and yield of HMW DNA normalized by tissue weight (nY) in each DNA extract. The average %HMW values for eight of the 10 species and the average nY values for five of the 10 species were significantly higher in extracts from EDTA-treated tissues compared to extracts from untreated frozen tissues. For all 10 species, we observed no significant decreases in average %HMW or nY values in extracts of EDTA-thawed tissues compared to those extracted directly from frozen tissues. In contrast, EtOH treatment did not significantly improve the average %HMW or nY values in extracts from tissues of nine of the 10 species when compared to extracts prepared directly from frozen tissues. Therefore, investigators may consider EDTA treatment as a simple method for improving HMW DNA recovery from frozen tissues.
冷冻保存是在组织样本中保存高分子量（HMW）DNA（>10 kb）的金标准。然而，冷冻组织通常在DNA提取之前或期间解冻，这可能导致大量DNA降解。在本研究中，我们将10种海洋物种（5种鱼类和5种无脊椎动物）先前冷冻的组织在防腐剂乙二胺四乙酸（EDTA，250 mM，pH 10）或乙醇（EtOH，95%）中解冻，并在4°C下于各自的防腐剂中过夜保存，然后进行DNA提取。然后，我们将这些提取物中HMW DNA的回收率与直接从冷冻组织制备的提取物进行比较。为了评估这些处理对HMW DNA回收率的影响，我们测定了每个DNA提取物中高分子量DNA的百分比（%HMW）和按组织重量标准化的HMW DNA产量（nY）。与未处理的冷冻组织提取物相比，来自EDTA处理组织的提取物中，10个物种中的8个物种的平均%HMW值和10个物种中的5个物种的平均nY值显著更高。对于所有10个物种，我们观察到与直接从冷冻组织提取的提取物相比，EDTA解冻组织提取物中的平均%HMW或nY值没有显著降低。相比之下，与直接从冷冻组织制备的提取物相比，乙醇处理并未显著提高10个物种中9个物种的组织提取物中的平均%HMW或nY值。因此，研究人员可以考虑将EDTA处理作为一种从冷冻组织中提高HMW DNA回收率的简单方法。
