Zheng Hao, Tao Yuan-Ping, Chen Feng-Qiu, Li Hui-Fen, Zhang Zhi-De, Zhou Xue-Xun, Yang Yuan, Zhou Wei-Ping
National Liver Tissue Bank, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China.
Key Laboratory of Signaling Regulation and Targeting Therapy of Liver Cancer (SMMU), Ministry of Education, Shanghai, China.
Biopreserv Biobank. 2019 Oct;17(5):425-432. doi: 10.1089/bio.2019.0003. Epub 2019 Apr 26.
High-throughput transcript sequencing plays an important role in the study of hepatocellular carcinoma (HCC) occurrence and development. High-quality biospecimens, especially high-quality RNA, are the most basic prerequisites for obtaining good transcript sequencing data. Our purpose was to explore the treatment conditions of ischemic tissue samples that can be used to obtain high-integrity RNA before freezing the samples in liquid nitrogen. Postoperative tumor tissues (T) and adjacent normal tissues (AN) from 50 HCC patients were randomly selected from May 5, 2017, to June 15, 2017. Postoperative tissue specimens from each HCC patient were stratified by tissue type (T or AN), ischemia time (minutes), and ischemia temperature (°C) into 16 groups: T-4°C-15 minutes, T-4°C-30 minutes, T-4°C-60 minutes, T-4°C-120 minutes, T-24°C-15 minutes, T-24°C-30 minutes, T-24°C-60 minutes, T-24°C-120 minutes, AN-4°C-15 minutes, AN-4°C-30 minutes, AN-4°C-60 minutes, AN-4°C-120 minutes, AN-24°C-15 minutes, AN-24°C-30 minutes, AN-24°C-60 minutes, and AN-24°C-120 minutes. RNA integrity was detected by RNA integrity number (RIN) and 1% agarose gel electrophoresis. At an ischemia temperature of 4°C and ischemia time of >30 minutes, the RIN of T began to decrease. RIN also gradually decreased in T at an ischemia temperature of 4°C and in both T and AN at an ischemia temperature of 24°C for ischemia times 15, 30, 60, and 120 minutes. For an ischemia time ≤15 minutes and ischemia temperature 4°C or 24°C, the RINs of T and AN were significantly different. Furthermore, at ischemia temperature 4°C and ischemia time 30 and 60 minutes or ischemia temperature 24°C and ischemia time 30 minutes, the RIN of T was higher compared with AN. However, there was no significant difference in RIN between T and AN under other treatment conditions. Tissue quality is adversely affected by ischemia time and ischemia temperature. Therefore, temporary ischemia time (≤15 minutes) before snap freezing is key for maintaining high-integrity RNA in HCC tissues.
高通量转录组测序在肝细胞癌（HCC）发生发展的研究中发挥着重要作用。高质量的生物样本，尤其是高质量的RNA，是获得良好转录组测序数据的最基本前提条件。我们的目的是探索缺血组织样本在液氮冷冻前可用于获取高完整性RNA的处理条件。从2017年5月5日至2017年6月15日，随机选取50例HCC患者的术后肿瘤组织（T）和癌旁正常组织（AN）。将每位HCC患者的术后组织标本按组织类型（T或AN）、缺血时间（分钟）和缺血温度（℃）分为16组：T-4℃-15分钟、T-4℃-30分钟、T-4℃-60分钟、T-4℃-120分钟、T-24℃-15分钟、T-24℃-30分钟、T-24℃-60分钟、T-24℃-120分钟、AN-4℃-15分钟、AN-4℃-30分钟、AN-4℃-60分钟、AN-4℃-120分钟、AN-24℃-15分钟、AN-24℃-30分钟、AN-24℃-60分钟和AN-24℃-120分钟。通过RNA完整性数值（RIN）和1%琼脂糖凝胶电泳检测RNA完整性。在缺血温度为4℃且缺血时间>30分钟时，T组的RIN开始下降。在缺血温度为4℃时，T组的RIN也逐渐下降；在缺血温度为24℃时，T组和AN组在缺血时间15、30、60和120分钟时RIN均逐渐下降。对于缺血时间≤15分钟且缺血温度为4℃或24℃，T组和AN组的RIN有显著差异。此外，在缺血温度为4℃且缺血时间为30和60分钟或缺血温度为24℃且缺血时间为30分钟时，T组的RIN高于AN组。然而，在其他处理条件下，T组和AN组之间的RIN无显著差异。缺血时间和缺血温度会对组织质量产生不利影响。因此，速冻前的短暂缺血时间（≤15分钟）是维持HCC组织中高完整性RNA的关键。
