Reference gene selection for quantitative real-time polymerase chain reaction analysis

• Main Authors: Z Nie, P Lu, X Chen, Q Wang, X Meng, S Lu, X Dong, K Chen
• Format: Article
• Language: English
• Published: University of Modena and Reggio Emilia 2017-04-01
• Series: Invertebrate Survival Journal
• Subjects: Bombyx mori; reference genes; BmNPV; qPCR; immune genes
• Online Access: http://www.isj.unimo.it/articoli/ISJ463.pdf
• ISSN: 1824-307X

Bombyx mori nucleopolyhedrovirus (BmNPV) is the most serious viral disease in silkworms. To investigate the mechanisms of the immune responses of B. mori to a BmNPV infection, a suitable reference gene (RG) is necessary for normalizing data when studying the expression of genes in BmNPV-infected silkworms or cells. Thus, quantitative real-time PCR polymerase chain reaction was used to compare the stability of expression of nine potential RGs, including the actin A3, translation initiation factor 3 (TIF-3), TIF-A4, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S RNA, 28S RNA, TATA-binding protein (TBP), ribosomal protein L3 (Rpl3), and α-tubulin genes, in silkworms infected with BmNPV. The results were analyzed by BestKeeper, geNorm, and NormFinder software. Overall, α-tubulin exhibited the most stable gene expression in BmNPV-infected silkworms, and this was verified by western blotting of the α-tubulin protein. Moreover, we detected the expression of some genes involved in the immune signaling pathways of silkworms after BmNPV infection using α-tubulin as an internal RG.