Page 29 - 《水产学报》2026年第2期

文章编号: 1000-0615(2026)02-029103-24 , 2026, 50(2): 029103

JOURNAL OF FISHERIES OF CHINA

DOI: 10.11964/jfc.20250615025

李双, 谷敏, 叶孝文, 等. 跨组织/性别系统评估曼氏无针乌贼内参基因的稳定性 [J]. 水产学报, 2026, 50(2): 029103.
Li S, Gu M, Ye X W, et al. Systematic evaluation of reference gene stability across tissues and sexes in cuttlefish (Sepiella
japonica) [J]. Journal of Fisheries of China, 2026, 50(2): 029103 (in Chinese).

Systematic evaluation of reference gene stability across tissues and

sexes in cuttlefish (Sepiella japonica)

LI Shuang , GU Min , YE Xiaowen , YE Bin , LIU Siyu ,
YU Jia , ZHOU Xu , CHI Changfeng *
(National and Provincial Joint Engineering Research Centre for Marine Germplasm Resources Exploration and Utilization,
School of Marine Science and Technology, Zhejiang Ocean University, Zhoushan 316022, China)

Abstract: Quantitative real-time PCR (qPCR) is widely used for gene expression analysis, but its accuracy critically depends
on stable internal reference genes for normalization. In marine invertebrates, especially non-model taxa such as cephalopods,
systematic evaluation of reference genes is limited, leading to potential bias. The cuttlefish Sepiella japonica is ecologically and
economically important in China, yet previous molecular studies have often relied on single unvalidated reference genes, which
may compromise data reliability. This study aimed to systematically evaluate the stability of five commonly used reference
genes (18S, ef-1α, ef-1γ, gapdh, and β-actin) across multiple tissues and sexes of S. japonica, and to identify the most suitable
reference genes and optimal number for qPCR normalization. Fifteen to sixteen tissue types were collected from ten healthy
adults (five males and five females). Total RNA was extracted, reverse-transcribed, and analyzed by qPCR. Gene stability was
assessed using four algorithms (geNorm, NormFinder, BestKeeper, and ΔC t ) integrated with RefFinder, and the optimal gene
number was determined using geNorm pairwise variation (V n/n+1 < 0.15). Four transcriptome-derived genes (creld2, cd109,
acy1, and miox) were used for validation. The C t values of the five genes ranged from 15.47 to 20.83. β-actin and gapdh
showed pronounced variability in expression stability among tissues and sexes, indicating their limited suitability for normaliza-
tion. 18S exhibited the highest expression (mean C t : 15.47-16.29) and lowest variability but displayed sex-biased expression,
whereas ef-1α and ef-1γ remained consistently stable across most tissues in both sexes, with ef-1α being the most robust and
showing no sex-related bias. Although specific rankings varied among tissues and sexes, the comprehensive results indicated
that ef-1α and ef-1γ possessed the highest overall stability, followed by 18S, while β-actin and gapdh were the least stable. The
final comprehensive rankings were ef-1γ > ef-1α > 18S > gapdh > β-actin (male) and ef-1α > ef-1γ > 18S > gapdh > β-actin
(female). geNorm analysis (V 2/3 < 0.15) indicated that two genes, mainly ef-1α and ef-1γ, were generally sufficient for reliable
normalization in most tissues. Validation confirmed that normalization using the stable ef-1α and ef-1γ accurately reflected the
expression differences among tissues, whereas β-actin and gapdh can bias or confound statistical analyses. ef-1α and ef-1γ are
identified as the most reliable reference gene combination for qPCR analysis in S. japonica, while 18S can serve as an auxiliary

收稿日期：2025-06-17 修回日期：2025-10-16
资助项目：国家自然科学基金 (32473136，42406102)；浙江省自然科学基金 (LTGN24C190005)
第一作者：李双，从事海洋头足类生殖调控以及环境应激机理研究，E-mail：lishuang@zjou.edu.cn；
谷敏，从事海洋动物免疫调控机制探究，E-mail：1435483833@qq.com
通信作者：迟长凤，从事海洋生物资源发掘与保护开发利用研究，E-mail：chicf@zjou.edu.cn
©《水产学报》编辑部（CC BY-NC-ND 4.0） Copyright © Editorial Office of Journal of Fisheries of China (CC BY-NC-ND 4.0)
中国水产学会主办 sponsored by China Society of Fisheries https://www.china-fishery.cn
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