Chen Yen-Po, Hu Chiung-Chih, Tsai Sujune, Wen Zhi-Hong, Lin Chiahsin
Department of Obstetrics and Gynecology, Kaohsiung Armed Forces General Hospital, Kaohsiung, 80284, Taiwan.
Department of Obstetrics and Gynecology, Tri-Service General Hospital, National Defense Medical Center, Taipei, 90055, Taiwan.
Sci Rep. 2025 Apr 1;15(1):11046. doi: 10.1038/s41598-025-95258-6.
Few studies have investigated the subcellular effects of low temperature on gene expression in shrimp and most other crustaceans. Before gene expression analysis is conducted, suitable housekeeping genes (HKGs) must be confirmed to account for differences in reverse transcription process efficiency among samples. Thus, this study aimed to verify five frequently used HKGs, namely 18S ribosomal RNA (18S rRNA), ATPase, histone 3, β-actin, and glyceraldehyde 3-phosphate dehydrogenase (gapdh) for use in experiments for assessing the molecular-scale effects of cryopreservation on coral banded shrimp (Stenopus hispidus) embryos. To conduct chilling studies, we subjected S. hispidus embryos to incubation at either 26 °C (control) or 5 °C for 0, 4, 8, 16, or 32 h. The software tools GeNorm, NormFinder, and Bestkeeper were employed to identify the most suitable HKG. GeNorm identified histone 3 and 18S rRNA as the most stable genes. By contrast, NormFinder determined that 18S rRNA is a stable gene for eye-formation and pre-hatch stage samples. Finally, Bestkeeper determined that gapdh and β-actin are the most suitable genes. This study is the first to identify suitable HKGs for studying shrimp embryos at low temperatures. Its findings can aid future research on evaluating the effects of cryopreservation on gene expression in crustaceans.
很少有研究调查低温对虾及大多数其他甲壳类动物基因表达的亚细胞效应。在进行基因表达分析之前，必须确认合适的看家基因（HKGs），以解释样本间逆转录过程效率的差异。因此，本研究旨在验证五个常用的看家基因，即18S核糖体RNA（18S rRNA）、ATP酶、组蛋白3、β-肌动蛋白和甘油醛-3-磷酸脱氢酶（gapdh），用于评估冷冻保存对条纹珊瑚虾（Stenopus hispidus）胚胎分子水平影响的实验。为了进行低温研究，我们将条纹珊瑚虾胚胎在26°C（对照）或5°C下孵育0、4、8、16或32小时。使用软件工具GeNorm、NormFinder和Bestkeeper来确定最合适的看家基因。GeNorm将组蛋白3和18S rRNA鉴定为最稳定的基因。相比之下，NormFinder确定18S rRNA是眼形成期和孵化前期样本的稳定基因。最后，Bestkeeper确定gapdh和β-肌动蛋白是最合适的基因。本研究首次确定了适合低温研究虾胚胎的看家基因。其研究结果有助于未来评估冷冻保存对甲壳类动物基因表达影响的研究。
