Validation of Reference Genes for Quantitative Real-Time PCR in Bovine PBMCs Transformed and Non-transformed by Theileria annulata.
- Author:
Hongxi ZHAO
1
;
Junlong LIU
;
Youquan LI
;
Congshan YANG
;
Shuaiyang ZHAO
;
Juan LIU
;
Aihong LIU
;
Guangyuan LIU
;
Hong YIN
;
Guiquan GUAN
;
Jianxun LUO
Author Information
- Publication Type:Original Article ; Research Support, Non-U.S. Gov't
- Keywords: Theileria annulata; quantitative real-time PCR (qPCR); reference gene; signaling pathway
- MeSH: Animals; B-Lymphocytes/parasitology; Cattle; Cell Line; Cells/*parasitology; Cells, Cultured; Gene Expression Profiling; Host-Parasite Interactions/*genetics; Real-Time Polymerase Chain Reaction/*veterinary; Reproducibility of Results; Signal Transduction/*genetics; Theileria annulata/physiology; Theileriasis/*physiopathology
- From:The Korean Journal of Parasitology 2016;54(1):39-46
- CountryRepublic of Korea
- Language:English
- Abstract: Theileria annulata is a tick-borne intracellular protozoan parasite that causes tropical theileriosis, a fatal bovine lymphoproliferative disease. The parasite predominantly invades bovine B lymphocytes and macrophages and induces host cell transformation by a mechanism that is not fully comprehended. Analysis of signaling pathways by quantitative real-time PCR (qPCR) could be a highly efficient means to understand this transformation mechanism. However, accurate analysis of qPCR data relies on selection of appropriate reference genes for normalization, yet few papers on T. annulata contain evidence of reference gene validation. We therefore used the geNorm and NormFinder programs to evaluate the stability of 5 candidate reference genes; 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ACTB (β-actin), PRKG1 (protein kinase cGMP-dependent, type I) and TATA box binding protein (TBP). The results showed that 18S rRNA was the reference gene most stably expressed in bovine PBMCs transformed and non-transformed with T. annulata, followed by GAPDH and TBP. While 18S rRNA and GAPDH were the best combination, these 2 genes were chosen as references to study signaling pathways involved in the transformation mechanism of T. annulata.
