Page 45 - 《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

V 2/3 V 3/4 V 4/5
0.50
0.45
0.40
pairwise variation/V 0.30 0.177 0.278 0.282 0.169 0.178 0.155 0.172 0.181
0.35

0.25
0.20
0.15
0.10 * * * * *
*
0.05 *
0
B OL H GI BH I ST C K L P WB SK T SP All
tissue
(a)

V 2/3 V 3/4 V 4/5
0.50
0.45
0.40 0.301
pairwise variation/V 0.30 0.173 0.240 0.158 0.233 0.279 0.191
0.35

0.25
0.20
0.15
0.10 * * * * * * *
*
0.05
0
B OL H GI BH I ST C K L P WB SK O NG ANG All
tissue
(b)
Fig. 10 Determination of the optimal number of reference genes for
normalization for male (a) and female (b) S. japonica
The dotted line represents the cut-off value of 0.15 for pairwise variation (V) analysis. When V n/n+1 < 0.15, n indicates the ideal number of reference
genes. The optimal number of reference genes for each group is marked with an asterisk. The arrows indicate the smallest V n/n+1 value when all V n/n+1 val-
ues greater than 0.15. B. brain, OL. optic lobe, H. heart, GI. gill, BH. branchial heart, I. intestine, ST stomach, C. caecus, K. kidney, L. liver, P. pancreas,
WB. white body, SK. skin, T. testis, SP. spermatophore, O. ovary, NG. nidamental gland, ANG. accessory nidamental gland, All. all tissues.

sequent qPCR study on reference gene stability, an in- In the gill and kidney, the previous evaluation of
depth bioinformatic mining was performed on the pre- reference gene stability indicated that, in both tissue-
existing gill and kidney transcriptome datasets of male specific and comprehensive analyses, ef-1α and ef-1γ
S. japonica from our research group. This analysis constituted the most stable combination of reference
identified four target genes (creld2, cd109, acy1, and genes in males, while gapdh and β-actin were the least
miox) that exhibited significantly differential read stable. Based on these results, we employed the two
counts between the two tissues. Specifically, the sets of reference genes separately for qPCR validation
expression levels of creld2 and cd109 were signific- in these two healthy tissues. After normalization, the
antly higher in the gill compared to the kidney, while expression trends of all four target genes were consist-
the inverse pattern was observed for acy1 and miox ent with the transcriptomic data under both normaliza-
(Fig. 11-a-d). tion strategies (Fig. 11-e-f). However, a critical differ-

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