Β-actin gene expression is variable among individuals and not suitable for normalizing mRNA levels in Portunus trituberculatus. Issue 81 (October 2018)

• Record Type: Journal Article
• Authors: Zhou, Su-Ming; Tao, Zhen; Shen, Chen; Qian, Dong; Wang, Chun-Lin; Zhou, Qi-Cun; Jin, Shan
• Is Part Of: Fish & shellfish immunology. Issue 81(2018)
• Page Start: 338
• Page End: 342
• Publication Date: 2018-10
• Subjects: Portunus trituberculatus -- Quantitative real-time PCR -- β-actin -- Reference gene; Fishes -- Immunology -- Periodicals; Shellfish -- Immunology -- Periodicals; Poissons -- Immunologie -- Périodiques; Crustacés -- Immunologie -- Périodiques; 571.9617
• DOI: 10.1016/j.fsi.2018.07.021
• Languages: English
• ISSNs: 1050-4648

Abstract

Abstract: The housekeeping gene encoding β-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 β-actin to normalize mRNA levels expression in many crustacean species is still object of debate. To date, it is still unclear if β-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 β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A, elongation factor 1-α (EF1-α), and 18 S ribosomal RNA (18 S rRNA) in the swimming crabs ( Portunus trituberculatu s) models. Our data showed that the β-actin gene expression varied significantly across individual swimming crab individuals in gills or hemocytes and the expression of 18 S rRNA, EF1-α, cyclophilin or GAPDH gene were relatively stable compared to that of β-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-α, compared to β-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. Highlights: β-actin gene expression varied significantly across individual swimming crab individuals. 18S rRNA was most stably expressed among tissues, followed by cyclophilin A and EF1-α in swimming crab. After WSSV infection, GAPDH and cyclophilin A were ranked as the most stable reference gene in gills and hemocytes, respectively.