Page 47 - 《水产学报》2026年第2期

2 期 李 双，等：跨组织/性别系统评估曼氏无针乌贼内参基因的稳定性 50 卷

Our findings are consistent with previous reports target genes.
in mammals and Drosophila melanogaster, which Nevertheless, in some female tissues (e.g., ovary,
reported sexual dimorphism in their expression levels nidamental gland) and in integrated datasets, even five
[2]
of commonly used reference genes (β-actin, 18S, and candidate genes did not meet the V n/n+1 <0.15 cutoff .
gapdh) [48-49] . Collectively, these results confirm that This highlights the profound tissue-specific complex-
many conventional reference genes are not univer- ity in finding a universal set of reference genes, even
sally stable, as their expression can vary substantially within the same sex. Given ongoing debate over the
across tissues, developmental stages, and experi- 0.15 threshold [2, 15, 55] , we advocate for combining cri-
mental conditions [50-52] , highlighting the need for sys- teria, such as selecting the set with the minimal V n/n+1
tematic validation in non-model organisms. value, particularly in non-model organisms like cuttle-
fish.
3.2 Potential of ef-1α and ef-1γ as robust nor-
Overall, ef-1α and ef-1γ exhibited the relatively
malization genes within each sex
strongest stability potential and can serve as reliable
Accurate normalization is fundamental for reli-
normalization genes within the same sex. However,
able qPCR analysis. Classic reference genes such as
their stability is not entirely uniform among tissues,
[10]
gapdh and β-actin are not universally stable, as
emphasizing that even top-ranking reference genes
[53]
shown in mouse gonads and Octopus vulgaris
should be validated for each tissue before use. Thus,
[54]
brain . Similarly, our multi-algorithm evaluation
the ef-1α/ef-1γ combination represents a robust start-
across male and female S. japonica tissues consist-
ing point rather than a universally invariant solution.
ently indicated that gapdh and β-actin were generally
unstable across most tissues, with only a few tissue- 3.3 Possible mechanisms underlying sex- and
specific exceptions. Notably, over 75% of prior S. tissue-specific differences in reference gene sta-
japonica expression studies relied solely on β-actin bility
(Tab. 1), reflecting a common practice in non-model
Although ef-1α and ef-1γ showed the greatest
organisms lacking systematic validation. Therefore,
overall stability, both gene stability patterns and rank-
future studies in this species should use β-actin and
ings varied across tissues and sexes. Three notable pat-
gapdh cautiously as normalization genes across tis-
terns emerged: ① stability differences between sexes
sues and sexes.
within the same tissues, ② variation among tissues
In contrast, ef-1α and ef-1γ (and, to a lesser
within a sex, and ③ generally higher variability in
extent, 18S) showed relatively high stability across
females.
multiple tissues within the same sex, although their
Sexual dimorphism is a major biological source
ranking varied among tissues. RefFinder ranking con-
of variation. Studies in mammals, Drosophila, and
sistently identified ef-1α/ef-1γ pair as top performers.
crustaceans have demonstrated that sex influences
and geNorm analysis showed that two genes (V 2/3 <
gene expression through hormonal regulation, tissues-
0.15) were sufficient for normalization in many tis-
specific activity, and chromosomal or cellular differ-
sues (e.g., male heart, female brain). Although certain
complex tissues like the female white body required ences [26, 27, 56-58] . Fluctuations in sex hormones and meta-
three genes (V 3/4 < 0.15), the ef-1α/ef-1γ pair still bolic states can modulate transcriptional and transla-
provided the most consistent results across sexes. tional efficiency, thereby affecting the expression of
Independent validation with transcriptome data fur- classical housekeeping genes such as β-actin and
ther confirmed that normalization with this pair repro- gapdh [57-60] . Consistently, 18S and β-actin in our data
duced transcriptome-derived differential expression exhibited pronounced sex-related expression differ-
patterns, whereas normalization with gapdh/β-actin ences in several tissues, directly influencing their sta-
led to loss of statistical significance in three of four bility rankings between males and females. These

中国水产学会主办 sponsored by China Society of Fisheries https://www.china-fishery.cn
19