Nakatsuji Nobuyoshi, Adachi Kohsuke, Morioka Katsuji
Faculty of Agriculture and Marine Science, Kochi University, Nankoku, Kochi, 783-8502, Japan.
The United Graduate School of Agricultural Sciences, Ehime University, Matsuyama, Ehime, 790-8566, Japan.
Fish Physiol Biochem. 2019 Apr;45(2):819-828. doi: 10.1007/s10695-018-0588-8. Epub 2018 Nov 19.
Recovering high-quality intact RNA from postmortem tissue is of major concern for gene expression studies. However, it is difficult to perform RNA extraction from aquacultured fish immediately after death, as rapid and accurate skills are needed for the procedure. The objective of this study was to quantitatively assess the integrity of total RNA extracted from muscle, liver, and digestive tract tissues of red seabream stored in ice as whole bodies, at a range of time points up to 10 days postmortem, using RNA integrity number (RIN) and quantitative PCR (qPCR). The RIN of total RNA in muscle remained over 8.0 for 5 days postmortem. The RINs in the liver and digestive tract were under 5.0 at 2 days postmortem. The mRNA levels of tissue inhibitor metalloproteinase 2 (TIMP-2) and β-actin, measured using qPCR in muscle, decreased to 87.8% at 1 day postmortem and to 45.5% at 2 days postmortem, from that at 0 days postmortem. In the liver and digestive tract, the mRNA levels were not significantly changed until 1 day postmortem. These results indicate that RNA, especially from fish muscle, can be maintained at high quality for several days postmortem solely by storing the fish body in ice.
从死后组织中获取高质量的完整RNA是基因表达研究的主要关注点。然而，在水产养殖鱼类死后立即进行RNA提取是困难的，因为该过程需要快速且准确的技术。本研究的目的是使用RNA完整性数值（RIN）和定量PCR（qPCR），在死后长达10天的一系列时间点，对以完整鱼体形式保存在冰中的红鲷肌肉、肝脏和消化道组织中提取的总RNA的完整性进行定量评估。肌肉中总RNA的RIN在死后5天内保持在8.0以上。肝脏和消化道中的RIN在死后2天时低于5.0。使用qPCR在肌肉中测量的组织金属蛋白酶抑制剂2（TIMP-2）和β-肌动蛋白的mRNA水平，相对于死后0天时，在死后1天时降至87.8%，在死后2天时降至45.5%。在肝脏和消化道中，mRNA水平直到死后1天没有显著变化。这些结果表明，仅通过将鱼体保存在冰中，RNA，尤其是来自鱼肌肉的RNA，在死后几天内可以保持高质量。
