Bai Mei, Liu Zi-Ling, Zhou Yu-Yu, Xu Qiu-Xuan, Liu Tong-Xian, Tian Hong-Gang
State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, China.
Key Laboratory of Integrated Pest Management on the Loess Plateau of Ministry of Agriculture, College of Plant Protection, Northwest A&F University, Yangling, China.
Pest Manag Sci. 2023 Jan;79(1):45-54. doi: 10.1002/ps.7171. Epub 2022 Sep 26.
A significant variation in RNA interference (RNAi) efficiency hinders further functional gene studies and pest control application in many insects. The available double-stranded RNA (dsRNA) molecules introduced into the target cells are regarded as the crucial factor for efficient RNAi response. However, numerous studies have only focused on dsRNA stability in vivo; it is uncertain whether different dsRNA storage conditions in vitro play a role in variable RNAi efficiency among insects.
A marker gene cardinal, which leads to white eyes when knocked-down in the red flour beetle Tribolium castaneum, was used to evaluate the effects of RNAi efficiency under different dsRNA storage conditions. We demonstrated that the dsRNA molecule is very stable under typical cryopreservation temperatures (-80 and -20 °C) within 180 days, and RNAi efficiency shows no significant differences under either low temperature. Unexpectedly, while dsRNA molecules were treated with multiple freeze-thaw cycles up to 50 times between -80/-20 °C and room temperature, we discovered that dsRNA integrity and RNAi efficiency were comparable with fresh dsRNA. Finally, when the stability of dsRNA was further measured under refrigerated storage conditions (4 °C), we surprisingly found that dsRNA is still stable within 180 days and can induce an efficient RNAi response as that of initial dsRNA.
Our results indicate that dsRNA is extraordinarily stable under various temperature storage conditions that did not significantly impact RNAi efficiency in vivo insects. © 2022 Society of Chemical Industry.
RNA干扰（RNAi）效率的显著差异阻碍了在许多昆虫中进一步开展功能基因研究和害虫防治应用。引入靶细胞的可用双链RNA（dsRNA）分子被视为高效RNAi反应的关键因素。然而，众多研究仅关注dsRNA在体内的稳定性；体外不同的dsRNA储存条件是否在昆虫间可变的RNAi效率中发挥作用尚不确定。
使用一个标记基因cardinal（在赤拟谷盗中敲低时会导致白眼）来评估不同dsRNA储存条件下的RNAi效率。我们证明dsRNA分子在典型的冷冻保存温度（-80和-20°C）下180天内非常稳定，并且在这两种低温下RNAi效率均无显著差异。出乎意料的是，当dsRNA分子在-80/-20°C和室温之间进行多达50次的多次冻融循环处理时，我们发现dsRNA完整性和RNAi效率与新鲜dsRNA相当。最后，当在冷藏条件（4°C）下进一步测量dsRNA的稳定性时，我们惊奇地发现dsRNA在180天内仍然稳定，并且能够像初始dsRNA一样诱导高效的RNAi反应。
我们的结果表明，dsRNA在各种温度储存条件下都非常稳定，这些条件不会对体内昆虫的RNAi效率产生显著影响。© 2022化学工业协会。
