title: Validation of Reference Genes for Quantitative Real-Time PCR in Bovine PBMCs Transformed and Non-transformed by Theileria annulata.
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.
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, People's Republic of China ; Agricultural College of Ningxia University, Yinchuan 750021, People's Republic of China.
authors.forename: Hongxi
authors.initials: H
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, People's Republic of China.
authors.forename: Junlong
authors.initials: J
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, People's Republic of China.
authors.forename: Youquan
authors.initials: Y
authors.lastname: Li
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, People's Republic of China.
authors.forename: Congshan
authors.initials: C
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, People's Republic of China.
authors.forename: Shuaiyang
authors.initials: S
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, People's Republic of China.
authors.forename: Juan
authors.initials: J
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, People's Republic of China.
authors.forename: Aihong
authors.initials: A
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, People's Republic of China.
authors.forename: Guangyuan
authors.initials: G
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, People's Republic of China ; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, People's Republic of China.
authors.forename: Hong
authors.initials: H
authors.lastname: Yin
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, People's Republic of China.
authors.forename: Guiquan
authors.initials: G
authors.lastname: Guan
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, People's Republic of China.
authors.forename: Jianxun
authors.initials: J
authors.lastname: Luo
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000469:parasitology; D002417:Cattle; D002460:Cell Line; D002477:Cells / Q000469:parasitology*; D002478:Cells, Cultured; D020869:Gene Expression Profiling; D006790:Host-Parasite Interactions / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary*; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000235:genetics*; D016796:Theileria annulata / Q000502:physiology; D013801:Theileriasis / Q000503:physiopathology*
keywords: Theileria annulata; quantitative real-time PCR (qPCR); reference gene; signaling pathway