Page 30 - 《水产学报》2026年第2期

2 期 水 产 学 报 50 卷

gene for within-sex comparisons. The use of β-actin or gapdh alone is not recommended. This study establishes a systematic
framework for selecting reliable reference genes in S. japonica, thereby facilitating robust qPCR normalization and providing a
foundation for future gene expression research in S. japonica and other cephalopods.
Key words: Sepiella japonica; reference genes; qPCR; expression stability; RefFinder
Corresponding author: CHI Changfeng. E-mail: chicf@zjou.edu.cn
Funding projects: National Natural Science Foundation of China (32473136, 42406102); Natural Science Foundation of Zheji-
ang Province (LTGN24C190005)

In studies of marine invertebrates, many spe- experimental systems.
[2]
cies —particularly those from understudied So far, various algorithms, including geNorm ,
[11]
[12]
taxa —still lack complete or high-quality reference NormFinder , BestKeeper , and the ΔC method [13]
t
genomes. This limitation severely restricts the applica- have been used to assess reference gene stability. Each
tion of gene-editing technologies such as CRISPR- algorithm employs distinct statistical approaches,
Cas9, thereby hindering in-depth exploration of gene which might lead to discrepancies. Therefore, a robust
functions. Furthermore, the advancement of gene func- validation strategy often involves combining multiple
tional studies is often constrained by insufficient fund- algorithms and determining the optimal number of ref-
[14]
ing and limited technical support. Consequently, quan- erence genes for accurate normalization . Identifying
tifying messenger RNA (mRNA) expression levels the optimal number of reference genes is crucial for
remains a crucial approach for studying gene function accurate normalization; too few lead to insufficient
and regulatory mechanisms. Among these methods, normalization, while too many inflate experimental
quantitative real-time PCR (qPCR) is widely utilized cost and effort [2, 15] .
for its high specificity and reliability in analyzing gene Cuttlefish, Sepiella japonica, an economically
expression changes in living organisms or cells under important cephalopod, has been cultured in China for
[1]
various experimental conditions . However, the years. As research interest in its molecular biology
accuracy and comparability of qPCR data are highly grows, accurate gene expression analysis has become
dependent on effective normalization. The most com- essential. Previous gene expression studies on this spe-
mon and recommended normalization strategy cies mainly relied on a single traditional control gene,
involves the use of stably expressed reference genes, β-actin. Recently, two or three reference genes have
often referred to as reference genes or housekeeping been gradually adopted to improve the reliability. In
[2]
genes . addition to β-actin, gapdh, tubulin, and ef-1γ have also
Ideally, an internal control gene should exhibit been included [16-22] . However, comprehensive studies
[3]
constant expression levels across conditions . evaluating the stability of these candidate genes across
However, numerous studies across diverse organisms diverse tissues and between sexes are still lacking.
have demonstrated that the expression stability of Given the known sexual dimorphism and complex tis-
[23]
commonly used reference genes can vary with tissue sue organization in cephalopods , a thorough valida-
type [3-5] , developmental stage [6-7] , health status [8-9] , and tion of reference genes across tissues and sexes is crit-
[5]
different environmental stimuli . Therefore, a single ical for reliable qPCR in this species (Tab. 1).
“universal” reference gene is rarely sufficient, and the This study aims to systematically evaluate the
inappropriate selection of internal controls can lead to expression stability of five commonly used candidate
inaccurate data normalization, misinterpretation of reference genes across diverse tissues from both male
gene expression patterns, and ultimately, erroneous and female S. japonica. The geNorm, NormFinder,
[10]
biological conclusions . Consequently, the system- BestKeeper, and the ΔC method were employed, fol-
t
atic validation of candidate reference genes is essen- lowed by a comprehensive ranking using RefFinder, to
tial for reliable gene expression analysis of different identify the most stable reference genes and determine

https://www.china-fishery.cn 中国水产学会主办 sponsored by China Society of Fisheries
2