Parasites, Hosts and Diseases

The Korea Society for Parasitology and Tropical Medicine (대한기생충학·열대의학회)
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Validation of Reference Genes for Quantitative Real-Time PCR in Bovine PBMCs Transformed and Non-transformed by Theileria annulata

  • Zhao, Hongxi (State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Liu, Junlong (State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Li, Youquan (State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Yang, Congshan (State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Zhao, Shuaiyang (State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Liu, Juan (State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Liu, Aihong (State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Liu, Guangyuan (State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Yin, Hong (State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Guan, Guiquan (State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Luo, Jianxun (State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences)
  • Received : 2015.02.04
  • Accepted : 2015.11.01
  • Published : 2016.02.29
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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 (${\beta}-actin$), 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.

Keywords

References

  1. Higuchi R, Fockler C, Dollinger G, Watson R. Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology 1993; 11: 1026-1030.
  2. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996; 6: 986-994. https://doi.org/10.1101/gr.6.10.986
  3. Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 2000; 25: 169-193. https://doi.org/10.1677/jme.0.0250169
  4. Lee PD, Sladek R, Greenwood CM, Hudson TJ. Control genes and variability: absence of ubiquitous reference transcripts in diverse mammalian expression studies. Genome Res 2002; 12: 292-297. https://doi.org/10.1101/gr.217802
  5. Huggett J, Dheda K, Bustin S, Zumla A. Real-time RT-PCR normalisation; strategies and considerations. Genes Immun 2005; 6: 279-284. https://doi.org/10.1038/sj.gene.6364190
  6. Radonic A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A. Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun 2004; 313: 856-862. https://doi.org/10.1016/j.bbrc.2003.11.177
  7. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 2008; 3: 1101-1108. https://doi.org/10.1038/nprot.2008.73
  8. Wang M, Wang Q, Zhang B. Evaluation and selection of reliable reference genes for gene expression under abiotic stress in cotton (Gossypium hirsutum L.). Gene 2013; 530: 44-50. https://doi.org/10.1016/j.gene.2013.07.084
  9. Yang CG, Wang XL, Tian J, Liu W, Wu F, Jiang M, Wen H. Evaluation of reference genes for quantitative real-time RT-PCR analysis of gene expression in Nile tilapia (Oreochromis niloticus). Gene 2013; 527: 183-192. https://doi.org/10.1016/j.gene.2013.06.013
  10. Dhar AK, Bowers RM, Licon KS, Veazey G, Read B. Validation of reference genes for quantitative measurement of immune gene expression in shrimp. Mol Immunol 2009; 46: 1688-1695. https://doi.org/10.1016/j.molimm.2009.02.020
  11. Zhang X, Ding L, Sandford AJ. Selection of reference genes for gene expression studies in human neutrophils by real-time PCR. BMC Mol Biol 2005; 6: 4. https://doi.org/10.1186/1471-2199-6-4
  12. Lossos IS, Czerwinski DK, Wechser MA, Levy R. Optimization of quantitative real-time RT-PCR parameters for the study of lymphoid malignancies. Leukemia 2003; 17: 789-795. https://doi.org/10.1038/sj.leu.2402880
  13. Schmittgen TD, Zakrajsek BA. Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J Biochem Bioph Meth 2000; 46: 69-81. https://doi.org/10.1016/S0165-022X(00)00129-9
  14. Tricarico C, Pinzani P, Bianchi S, Paglierani M, Distante V, Pazzagli M, Bustin SA, Orlando C. Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies. Anal Biochem 2002; 309: 293-300. https://doi.org/10.1016/S0003-2697(02)00311-1
  15. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002; 3: RESEARCH0034.
  16. Andersen CL, Jensen JL, Orntoft TF. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 2004; 64: 5245-5250. https://doi.org/10.1158/0008-5472.CAN-04-0496
  17. Chaussepied M, Langsley G. Theileria transformation of bovine leukocytes: a parasite model for the study of lymphoproliferation. Res Immunol 1996; 147: 127-138. https://doi.org/10.1016/0923-2494(96)83165-8
  18. Li Y, Liu Z, Yang J, Chen Z, Guan G, Niu Q, Zhang X, Luo J, Yin H. Infection of small ruminants and their red blood cells with Theileria annulata schizonts. Exp Parasitol 2014; 137: 21-24. https://doi.org/10.1016/j.exppara.2013.11.010
  19. Spooner RL, Innes EA, Glass EJ, Brown CG. Theileria annulata and T. parva infect and transform different bovine mononuclear cells. Immunology 1989; 66: 284-288.
  20. Ahmed JS, Rintelen M, Schein E, Williams RO, Dobbelaer, D. Effect of buparvaquone on the expression of interleukin 2 receptors in Theileria annulata-infected cells. Parasitol Res 1992; 78: 285-290. https://doi.org/10.1007/BF00937085
  21. Brown CG, Stagg DA, Purnell RE, Kanhai GK, Payne RC. Letter: Infection and transformation of bovine lymphoid cells in vitro by infective particles of Theileria parva. Nature 1973; 245: 101-103. https://doi.org/10.1038/245101a0
  22. Yin H, Luo J, Schnittger L, Lu B, Beyer D, Ma M, Guan G, Bai Q, Lu C, Ahmed J. Phylogenetic analysis of Theileria species transmitted by Haemaphysalis qinghaiensis. Parasitol Res 2004; 92: 36-42. https://doi.org/10.1007/s00436-003-0900-z
  23. Yamaguchi T, Yamanaka M, Ikehara S, Kida K, Kuboki N, Mizuno D, Yokoyama N, Narimatsu H, Ikehara Y. Generation of IFN-gamma-producing cells that recognize the major piroplasm surface protein in Theileria orientalis-infected bovines. Vet Parasitol 2010; 171: 207-215. https://doi.org/10.1016/j.vetpar.2010.03.038
  24. Kubista M, Andrade JM, Bengtsson M, Forootan A, Jonak J, Lind K, Sindelka R, Sjoback R, Sjogreen B, Strombom L, Stahlber, A, Zoric N. The real-time polymerase chain reaction. Mol Aspects Med 2006; 27: 95-125. https://doi.org/10.1016/j.mam.2005.12.007
  25. Winer J, Jung CK, Shackel I, Williams PM. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem 1999; 270: 41-49. https://doi.org/10.1006/abio.1999.4085
  26. Ginzinger DG. Gene quantification using real-time quantitative PCR:An emerging technology hits the mainstream. Exp Hematol 2002; 30: 503-512. https://doi.org/10.1016/S0301-472X(02)00806-8
  27. Tong Z, Gao Z, Wang F, Zhou J, Zhang Z. Selection of reliable reference genes for gene expression studies in peach using real-time PCR. BMC Mol Biol 2009; 10: 71. https://doi.org/10.1186/1471-2199-10-71
  28. Kuchipudi SV, Tellabati M, Nelli RK, White GA, Perez BB, Sebastian S, Slomka MJ, Brookes SM, Brown IH, Dunham SP, Chang KC.18S rRNA is a reliable normalisation gene for real time PCR based on influenza virus infected cells. Virol J 2012; 9: 230. https://doi.org/10.1186/1743-422X-9-230
  29. Khurshid N, Agarwal V, Iyengar S, Expression of mu- and delta-opioid receptors in song control regions of adult male zebra finches (Taenopygia guttata). J Chem Neuroanat 2009; 37: 158-169. https://doi.org/10.1016/j.jchemneu.2008.12.001
  30. Mukai M, Replogle K, Drnevich J, Wang G, Wacker D, Band M, Clayton DF, Wingfield JC. Seasonal differences of gene expression profiles in song sparrow (Melospiza melodia) hypothalamus in relation to territorial aggression. PLoS One 2009; 4: e8182. https://doi.org/10.1371/journal.pone.0008182
  31. Dheda K, Huggett JF, Chang JS, Kim LU, Bustin SA, Johnson MA, Rook GA, Zumla A. The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization. Anal Biochem 2005; 344: 141-143. https://doi.org/10.1016/j.ab.2005.05.022

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