Korean J Parasitol.
2016 Feb;54(1):39-46. 10.3347/kjp.2016.54.1.39.
Validation of Reference Genes for Quantitative Real-Time PCR in Bovine PBMCs Transformed and Non-transformed by Theileria annulata
- Affiliations
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- 1State 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. guanguiquan@c
- 2Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, People's Republic of China.
- 3Agricultural College of Ningxia University, Yinchuan 750021, People's Republic of China.
- KMID: 2374015
- DOI: http://doi.org/10.3347/kjp.2016.54.1.39
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.
MeSH Terms
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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
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