Ji Xiaoli, Wang Min, Li Lingling, Chen Fang, Zhang Yanyang, Li Qian, Zhou Junmei
1 Department of Central Laboratory, Shanghai Children's Hospital, Shanghai Jiao Tong University , Shanghai, China .
2 Department of Urology, Shanghai Children's Hospital, Shanghai Jiao Tong University , Shanghai, China .
Biopreserv Biobank. 2017 Oct;15(5):475-483. doi: 10.1089/bio.2017.0064. Epub 2017 Sep 20.
High-quality biosamples are valuable resources for biomedical research. However, some tissues are stored without being sectioned into small aliquots and have to undergo repeated freeze-thaw cycles throughout prolonged experimentation. Little is known regarding the effects of repeated freeze-thaw cycles on the quality of biomolecules in tissues. The aim of this study was to evaluate the impact of repeated freeze-thaw (at room temperature or on ice) cycles on biomolecules and gene expression in four different types of tissues. Each fresh tissue was sectioned into seven aliquots and snap-frozen before undergoing repeated freeze-thaw cycles at room temperature or on ice. Biomolecules were extracted and analyzed. Both relative and absolute quantification were used to detect the changes in gene expression. The results indicated that the impact of repeated freeze-thaw cycles on RNA integrity varied by tissue type. Gene expression, including the housekeeping gene, was affected in RNA-degraded samples according to absolute quantification rather than relative quantification. Furthermore, our results suggest that thawing on ice could protect RNA integrity compared with thawing at room temperature. No obvious degradation of protein or DNA was observed with repeated freeze-thaw cycles either at room temperature or on ice. This research provides ample evidence for the necessity of sectioning fresh tissues into small aliquots before snap-freezing, thus avoiding degradation of RNA and alteration of gene expression resulting from repeated freeze-thaw cycles. For frozen tissue samples that were already in storage and had to be used repeatedly during their lifecycle, thawing on ice or sectioned at ultralow temperature is recommended.
高质量的生物样本是生物医学研究的宝贵资源。然而，一些组织在储存时没有被切成小份，并且在长时间的实验过程中必须经历反复的冻融循环。关于反复冻融循环对组织中生物分子质量的影响，人们了解甚少。本研究的目的是评估反复冻融（在室温或冰上）循环对四种不同类型组织中生物分子和基因表达的影响。将每个新鲜组织切成七份，速冻后在室温或冰上进行反复冻融循环。提取并分析生物分子。使用相对定量和绝对定量来检测基因表达的变化。结果表明，反复冻融循环对RNA完整性的影响因组织类型而异。根据绝对定量而非相对定量，包括管家基因在内的基因表达在RNA降解的样本中受到影响。此外，我们的结果表明，与在室温下解冻相比，在冰上解冻可以保护RNA的完整性。在室温或冰上进行反复冻融循环时，未观察到蛋白质或DNA有明显降解。本研究为在速冻前将新鲜组织切成小份提供了充分的证据，从而避免了由于反复冻融循环导致的RNA降解和基因表达改变。对于已经储存且在其生命周期中必须反复使用的冷冻组织样本，建议在冰上解冻或在超低温下切片。
