Page 38 - 《水产学报》2026年第2期

2 期 水 产 学 报 50 卷

being identified as the least stable gene in six tissue ing aligned with the tissue-specific quantification res-
types. Unlike in males, 18S in females demonstrated ults obtained for females but exhibited a minor dis-
enhanced stability, consistently ranking among the top crepancy when compared to the findings from male S.
three genes in over half of the tissues and as the most japonica.
stable gene in seven tissues. Notably, ef-1γ, gapdh, and Despite a general similarity in overall gene sta-
18S were unsuitable as reference genes in the caecus bility patterns, distinct tissue-specific stability rank-
(Fig. 4-h), and gapdh further failed to meet stability ings for the five candidate genes were observed
criteria (M > 1.5) in the ovary (Fig. 4-n). The integ- between male and female S. japonica. For instance, in
rated analysis revealed a consistent ranking pattern of the male brain, the stability ranking was ef-1α > 18S >
candidate genes in females, namely ef-1α > ef-1γ > ef-1γ > gapdh > β-actin, while it was 18S > β-actin >
18S > gapdh > β-actin. However, when all tissues ef-1γ > ef-1α > gapdh in the female brain.
were analyzed as a single sample (Fig. 4-q), gapdh and 2.5 BestKeeper analysis
β-actin were not recommended for use.
The third tool used to evaluate the stability of
2.4 NormFinder analysis reference genes was BestKeeper. This program

Gene stability was further evaluated using the provides various output parameters, among which the
NormFinder algorithm. According to the algorithm, it standard deviation (SD) is a key determinant of gene
stability. As with other algorithms, lower SD values
provides the reference gene with the highest stable
[11]
expression directly , where lower values indicate indicate greater stability. Notably, as documented in
greater stability. certain publications, only reference genes with an SD
In male S. japonica, ef-1γ consistently ranked as value less than 1.0 are considered acceptable.
the most stable gene based on individual tissue quanti- BestKeeper assessments in male S. japonica
fication, exhibiting low stability values in ten out of revealed a stability pattern that differed slightly from
fifteen tissues, specifically: the optic lobe, stomach, those observed with geNorm and NormFinder (Fig. 6-
caecus, kidney, liver, pancreas, white body, skin, a). In this analysis, 18S performed best across most tis-
testis, and spermatophore (Fig. 5-a,). Following ef-1γ, sues, followed by ef-1γ and ef-1α. Applying the stabil-
ef-1α demonstrated superior stability across a more ity criterion of SD < 1.0, all five genes were identified
extensive range of tissues. Among the remaining three as the shared suitable genes only in three tissues,
genes, 18S showed greater stability than gapdh and β- including the heart, branchial heart, and liver. Further-
actin. These tissue-specific findings were largely cor- more, 18S was found to be an acceptable reference con-
roborated by the integrated analysis, with the excep-
trol in thirteen tissues, while ef-1γ and gapdh were suit-
tion of ef-1α ranking higher than ef-1γ in this compre-
able in nine tissues, and ef-1α in seven. Notably, in the
hensive assessment.
stomach, caecus, kidney, and testis, 18S was the only
In females, the top three stable genes in most tis-
acceptable control gene. No gene had an SD < 1 in the
sues were consistent with in males, namely ef-1α, ef-
skin, although 18S (SD = 1.17) and β-actin (SD = 1.21)
1γ, and 18S, although their precise ranking varied
exhibited the closest values to this cutoff. The integ-
across tissues (Fig. 5-b). Nevertheless, the gene stabil-
rated analysis revealed that the SD values for all genes
ity pattern derived from the tissue-specific quantifica-
were greater than 1.5, with 18S (SD = 1.06) and ef-1γ
tions revealed that ef-1α outperformed ef-1γ, with ef-
(SD = 1.25) ranking as the top two, followed by ef-1α
1α emerging as the most preferred gene in seven dis-
tinct tissues. Conversely, the stability of gapdh and β- and gapdh. β-actin consistently ranked last.
actin consistently lagged behind, ranking as the two In female S. japonica, the overall stability of five
least stable genes. The integrated analysis (Fig. 5-b, all genes across tissues was better than that of males (Fig.
tissues) detected the rank order as follows: ef-1α > ef- 6-b). This is evidenced by all five genes being identi-
1γ > 18S > gapdh > β-actin. This comprehensive rank- fied as the shared suitable reference genes in over half

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