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2 期 水 产 学 报 50 卷

findings reinforce that sex-linked transcriptional bias females when no cross-sex invariant reference exists.
represents a key contributor to reference-gene instabil-
4 CONCLUSION
ity [48-49] .
When no pronounced sex difference is evident— In conclusion, this study systematically evaluated
either among tissues within one sex or between the stability of five conventional reference genes in
sexes with similar expression levels —individual diverse tissues of both male and female S. japonica.
variability becomes a major source of stability vari- Among all candidates, the commonly used reference
ation. The C range reflects expression consistency genes β-actin and gapdh showed pronounced variabil-
t
across individuals: a wider range indicates lower stabil- ity in expression stability among tissues and between
ity. For instance, ef-1α displayed comparable mean sexes, underscoring their limited suitability for normal-
expression levels in male and female pancreas but the ization. 18S exhibited the highest expression and low-
boadest C range in males, resulting in lower stability est variability but displayed sex-biased expression,
t
relative to β-actin and ef-1γ. In contrast, ef-1α in whereas ef-1α and ef-1γ remained consistently stable in
females showed the narrowest C variation and hence most tissues of both sexes, with ef-1α being the most
t
the highest stability. Moreover, stability rankings robust. Multi-algorithm analysis confirmed that ef-1α
depend not only on the variability of each gene but also and ef-1γ showed the greatest overall stability followed
on that of co-analyzed genes and the weighting by 18S, while β-actin and gapdh were the least stable.
strategies of algorithms such as RefFinder, which integ- geNorm pairwise variation analysis (V 2/3 < 0.15) indic-
rates geNorm, NormFinder, and BestKeeper [2, 11-12, 14] . ated that two genes, mainly ef-1α and ef-1γ, are gener-
This algorithmic difference may contribute to apparent ally sufficient for reliable normalization in most tissues.
reversals of stability between sexes or tissues, as they Validation using transcriptome-derived target genes
differ in how they account for C standard deviation, (creld2, cd109, acy1, miox) confirmed that stable refer-
t
intra-group consistency, and inter-gene correlation, ences such as ef-1α and ef-1γ accurately reflect the
even subtle data differences may yield sex-and tissue- expression differences among tissues, whereas unstable
dependent ranking shifts. references such as β-actin and gapdh can bias or con-
Interestingly, females consistently showed greater found statistical analyses. Nevertheless, variations in
variation in gene stability, a pattern also reported in stability patterns and rankings among tissues and
[53]
mouse models . While the underlying cause remains between sexes were also observed, likely caused by dif-
ferences in cellular composition, metabolic activity, and
uncertain, potential factors include greater physiolo-
sex-related physiological regulation, emphasizing the
gical heterogeneity linked to reproductive cycling, fluc-
tuating endocrine states [61-62] , and variable cellular com- need for tissue- and sex-specific validation. Overall,
these findings establish a systematic framework for
position in female-specific tissues such as the nida-
selecting reliable reference genes in S. japonica, thereby
mental gland and accessory nidamental gland. Future
facilitating robust qPCR normalization and providing a
studies should address these factors by sampling indi-
foundation for future gene expression research in this
viduals at defined reproductive stages and combining
species.
transcriptomic or proteomic analyses to better under-
stand sex-specific transcriptional regulation.
（作者声明本文无利益冲突）
Taken together, biological and analytical factors
contribute to sex- and tissue-specific variability in ref- 参考文献 (References)：
erence gene stability. For reliable qPCR normalization, [ 1 ] Smith C J, Osborn A M. Advantages and limitations of quantit-
reference gene stability should be assessed separately ative PCR (Q-PCR)-based approaches in microbial ecology[J].
for each tissue and sex, and the most stable genes FEMS Microbiology Ecology, 2009, 67(1): 6-20.
should be selected independently for males and [ 2 ] Vandesompele J, De Preter K, Pattyn F, et al. Accurate normal-

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