Yu Keke, Xing Jie, Zhang Jie, Zhao Ruiying, Zhang Ye, Zhao Lanxiang
Department of Pathology, Shanghai Chest Hospital, Shanghai JiaoTong University, West Huaihai Road, No. 241, Building 2, Room 4B, Shanghai, 230000, China.
Department of Biaobank, Shanghai Chest Hospital, Shanghai JiaoTong University, Shanghai, 230000, China.
Cell Tissue Bank. 2017 Sep;18(3):433-440. doi: 10.1007/s10561-016-9600-7. Epub 2017 Jun 1.
RNA degradation is a major problem in tissue banking. We explored the effect of thawing flash-frozen biospecimens on the quality and integrity of RNA for genetic testing as well as for other cancer research studies. The histological quality of the frozen tumor sections was evaluated by using hematoxylin and eosin staining. RNA extraction from 60 lung cancer tissue samples subjected to various freeze/thaw cycles was performed using the RNeasy Plus isolation kit. RNA integrity was assessed by using an Agilent bioanalyzer to obtain RNA integrity numbers (RIN). Furthermore, RNA from different groups was used for fluorescence Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (EML4-ALK) fusion gene mutation to verify whether it can be used for research or clinical testing. Highly variable RIN values were observed among the samples, which showed no correlation with the number of freeze/thaw cycles conducted. However, after 3 freeze/thaw cycles (each thaw event lasted for 10 min), an increasing number of changes in peak intensity in RINs were observed. After 5 freeze/thaw cycles, RNA integrity decreased to approximately 35%. After 3 freeze/thaw cycles, the RNA could still be used for RT-PCR analysis of EML4-ALK fusion gene mutations; whereas those subjected to 5 freeze/thaw cycles could not. Limited (<3) freeze/thaw cycles did not adversely affect the quality of RNA extracted from tumor tissues and subsequent RT-PCR analysis. Our data could be utilized in the establishment of a standardized procedure for tissue biospecimen collection and storage.
RNA降解是组织库中的一个主要问题。我们探讨了解冻速冻生物样本对用于基因检测以及其他癌症研究的RNA质量和完整性的影响。通过苏木精和伊红染色评估冷冻肿瘤切片的组织学质量。使用RNeasy Plus分离试剂盒从60个经历不同冻融循环的肺癌组织样本中提取RNA。使用安捷伦生物分析仪评估RNA完整性以获得RNA完整性数值（RIN）。此外，将不同组的RNA用于棘皮动物微管相关蛋白样4和间变性淋巴瘤激酶（EML4-ALK）融合基因突变的荧光逆转录-聚合酶链反应（RT-PCR）分析，以验证其是否可用于研究或临床检测。样本中观察到高度可变的RIN值，其与进行的冻融循环次数无关。然而，在3次冻融循环（每次解冻持续10分钟）后，观察到RIN中峰值强度的变化数量增加。在5次冻融循环后，RNA完整性降至约35%。在3次冻融循环后，RNA仍可用于EML4-ALK融合基因突变的RT-PCR分析；而经历5次冻融循环的RNA则不能。有限（<3）的冻融循环不会对从肿瘤组织中提取的RNA质量及后续RT-PCR分析产生不利影响。我们的数据可用于建立组织生物样本采集和储存的标准化程序。
