期刊
FISH & SHELLFISH IMMUNOLOGY
卷 81, 期 -, 页码 338-342出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.fsi.2018.07.021
关键词
Portunus trituberculatus; Quantitative real-time PCR; beta-actin; Reference gene
资金
- Natural Science Foundation of Zhejiang Province [LQ16C190001]
- National Natural Science Foundation of China [41476124]
- China Agriculture Research System-48 [CARS-48]
- Major projects of Ningbo [2017C110007]
- K.C.Wong Magna Fund in Ningbo University
The housekeeping gene encoding 13-actin appears to be the most widely-used internal reference for gene expression studies in experimental animals or their cell lines. However, the effectiveness of beta-actin to normalize mRNA levels expression in many crustacean species is still object of debate. To date, it is still unclear if beta-actin is suitable to be utilized as the internal reference in qualitative real-time gene expression study in crab species. To address this concern, we evaluated 5 candidate reference genes encoding beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A, elongation factor 1-alpha (EF1-alpha), and 18 S ribosomal RNA (18 S rRNA) in the swimming crabs (Portunus trituberculatus) models. Our data showed that the beta-actin gene expression varied significantly across individual swimming crab individuals in gills or hemocytes and the expression of 18 S rRNA, EF1-alpha, cyclophilin or GAPDH gene were relatively stable compared to that of beta-actin. Moreover, the expression stability of the reference genes among different tissues in normal crabs or after WSSV challenge was also tested by geNorm and NormFinder software. Among tissues, 18 S rRNA was most stably expressed in different tissues, followed by cyclophilin A and EF1-alpha, compared to beta-actin and GAPDH. Upon to viral simulation, GAPDH was found to be the most stable internal control gene in gills and cyclophilin A was ranked as the most stable gene in hemocytes.
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