Page 46 - 《水产学报》2026年第2期

2 期 水 产 学 报 50 卷

creld2 cd109 acy1 miox
20 000 5 000 000 8 000 8 000
18 000 4 500 000 7 000
16 000 4 000 000
14 000 3 500 000 6 000 6 000
5 000
no. of reads 10 000 no. of reads 2 500 000 no. of reads 4 000 no. of reads 4 000
3 000 000
12 000
500
200
100
400
80
150
300
100
200 60 2 000
40
50 100 20
0 0 0 0
GI K GI K GI K GI K
tissue tissue tissue tissue
(a) (b) (c) (d)
6 8 6 *
relative mRNA expression 4 *** ** ** ** relative mRNA expression 4 2
2

0 0
GI K GI K GI K GI K GI K GI K GI K GI K
creld2 cd109 acy1 miox creld2 cd109 acy1 miox
normalized to ef-1a and ef-1γ normalized to gapdh and β-actin
(e) (f)
Fig. 11 Tissue-specific transcript abundance and normalized relative mRNA expression four genes in S. japonica
GI, gill. K, kidney, n = 4. Statistical significance between male and female groups was performed using unpaired two-tailed t-test.
ence emerged in statistical significance: when normal- dependent variability of reference genes in S. japon-
ized using ef-1α and ef-1γ, all four genes showed sig- ica. Among the genes examined, 18S was generally
nificant expression differences between tissues (Fig. the most abundant and least variable, consistent with
11-e), whereas only acy1 reached statistical signific- its role as a ribosomal component. However, signific-
ance when normalized with gapdh and β-actin (Fig. 11-
ant sex-biased differences were detected in neural, car-
f). These findings demonstrate that the stability of ref-
diac, and digestive tissues. β-actin and gapdh exhib-
erence genes critically influences statistical outcomes
ited broader tissues-dependent fluctuations, particu-
and further substantiate our earlier conclusions.
larly in reproductive organs, with β-actin showing pro-
3 DISCUSSION nounced sex-biased expression in several tissues,
indicating transcriptional instability. In contrast,
The selection of stable reference genes is critical
elongation factor genes demonstrated higher stability
for accurate normalization in qPCR studies, particu-
across tissues, with ef-1α emerging as the most robust.
larly in non-model organisms. Here, the stability of
It showed no sex-related differences in any examined
five conventional internal reference genes (18S, ef-1α,
tissue and exhibited only slight overall variability, sup-
ef-1γ, gapdh, and β-actin) was systematically assessed
porting its robustness for cross-sex normalization.
using four distinct algorithms across multiple tissues
Although not as universally stable as ef-1α, ef-1γ still
of male and female cuttlefish S. japonica.
demonstrated less expression variability relative to tra-
3.1 Expression stability of conventional refer-
ditional reference genes, but its significant sex-biased
ence genes in cuttlefish
expression in the optic lobe, stomach, pancreas, and
Comparative analysis revealed tissue- and sex- white body constrains its applicability in these tissues.

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