Page 31 - 《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

Tab. 1 References list of β-actin as an internal reference gene in S. japonica (partial)
internal control gene internal control gene
seq. source seq. source
(accession no.) (accession no.)
1 β-actin (PV684488)* [24] 16 β-actin (PV684488) [39]
2 β-actin (PV684488) [25] 17 β-actin (JN564496.1) [40]
3 β-actin (PV684488) [26] 18 β-actin (JN564496.1) [41]
4 β-actin (JN564496.1) [27] 19 β-actin (PV684488) [42]
5 β-actin (PV684488) [28] 20 β-actin (PV684488) [43]
6 β-actin (PV684488) [29] 21 β-actin (Unknown) [44]
7 β-actin (PV684488) [30] 22 β-actin (PV684488) [45]
8 β-actin (PV684488) [31] 23 β-actin (PV684488) [46]
9 β-actin (JN564496.1) [32] 24 gapdh, tuba, β-actin (PV684488) [16]
10 β-actin (PV684488) [33] 25 gapdh, tuba, β-actin (PV684488) [17]
11 β-actin (PV684488) [34] 26 gapdh, β-actin (PV684488) [18]
12 β-actin (JN564496.1) [35] 27 gapdh, tuba, β-actin (Unknow) [19]
13 β-actin (JN564496.1) [36] 28 gapdh, β-actin (PV684488) [20]
14 β-actin (PV684488) [37] 29 tubulin, β-actin (PV684488) [21]
15 β-actin (PV684488) [38] 30 gapdh, ef-1γ, β-actin (PV684488) [22]
Notes: Only display the accession number of β-actin. * denotes a sequence recently submitted in this paper.

the optimal number of genes required for accurate nor- Zhejiang Ocean University.
malization in various tissue contexts. This research
1.2 Total RNA extraction and cDNA synthesis
will provide essential guidelines for gene expression
Total RNA of each sample was isolated using the
studies in S. japonica, enhancing our understanding of
its molecular biology and facilitating future research in TRIzol total RNA kit (TaKaRa, Japan). RNA concen-
cephalopod biology. tration and purity were measured using the Nano Drop
spectrophotometer (Thermo Fisher, USA). 1 μg RNA
1 MATERIAL AND METHODS was used for cDNA synthesis using the RT mix with
DNase (All-in-One) kit (UElandy, China). The 20 μL
1.1 Collection of S. japonica tissues
volume reaction system containing 4.0 μL 5×RT All-
Ten healthy S. japonica (five females and five in-One Mix, 1.0 μL DNase, 1.0 μg RNA and DNase/
males) were obtained from the breeding base of RNase-free water to a final volume of 20 μL. Reverse
Xishan Island, Zhoushan City, Zhejiang Province. The transcription was performed on a thermal cycler (Bio-
mean body length and weight were (57.16 ± 7.71) cm Rad, USA) under the following conditions: 2 min at
and (7.76 ± 0.29) g, respectively. The gill, branchial 37℃, 15 min at 55℃ and 5 min at 85℃. The cDNA
heart, kidney, heart, pancreas, liver, skin, gonad-asso- product was stored at −20℃ until use.
ciated tissues (ovary, nidamental gland, accessory
1.3 Reference gene selection and primer design
nidamental gland, testis, spermatophore), brain, white
body, optic lobe, intestine, stomach, and caecus were Five candidate reference genes were selected in
dissected and kept in 1 mL RNA store (Cwbiotech, this study: elongation factor 1 alpha (ef-1α), elonga-
China) immediately. A total of 15 and 16 tissues in tion factor 1 gamma (ef-1γ), glyceraldehyde-3-phos-
males and females were collected, respectively. All phate dehydrogenase (gapdh), beta-actin (β-actin), and
samples were stored at −80 ℃ until RNA extraction. 18S ribosomal RNA (18S). Basic information on these
All experimental protocols were approved by the Insti- candidate genes was listed in Tab. 2. Primers were
tutional Animal Care and Use Committee (IACUC) of designed using software Primer Premier 5.0 version.

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