Asian-Australasian Journal of Animal Sciences

Asian Australasian Association of Animal Production Societies (아세아태평양축산학회)
권호 표지
DOI QR코드

DOI QR Code

Selection of Reference Genes for Gene Expression Studies in Porcine Whole Blood and Peripheral Blood Mononuclear Cells under Polyinosinic:Polycytidylic Acid Stimulation

  • Wang, Jiying (Shandong Provincial Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences) ;
  • Wang, Yanping (Shandong Provincial Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences) ;
  • Wang, Huaizhong (Shandong Provincial Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences) ;
  • Hao, Xiaojing (Qingdao Institute of Animal Science and Veterinary Medicine) ;
  • Wu, Ying (Shandong Provincial Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences) ;
  • Guo, Jianfeng (Shandong Provincial Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences)
  • Received : 2013.08.05
  • Accepted : 2013.11.20
  • Published : 2014.04.01
Download PDF
⟨ Previous Next ⟩

Abstract

Investigating gene expression of immune cells of whole blood or peripheral blood mononuclear cells (PBMC) under polyinosinic:polycytidylic acid (poly I:C) stimulation is valuable for understanding the immune response of organism to RNA viruses. Quantitative real-time PCR (qRT-PCR) is a standard method for quantification of gene expression studies. However, the reliability of qRT-PCR data critically depends on proper selection of reference genes. In the study, using two different analysis programs, geNorm and NormFinder, we systematically evaluated the gene expression stability of six candidate reference genes (GAPDH, ACTB, B2M, RPL4, TBP, and PPIA) in samples of whole blood and PBMC with or without poly I:C stimulation. Generally, the six candidate genes performed a similar trend of expression stability in the samples of whole blood and PBMC, but more stably expressed in whole blood than in PBMC. geNorm ranked B2M and PPIA as the best combination for gene expression normalization, while according to NormFinder, TBP was ranked as the most stable reference gene, followed by B2M and PPIA. Comprehensively considering the results from the two programs, we recommended using the geometric mean of the three genes, TBP, PPIA and B2M, to normalize the gene expression of whole blood and PBMC with poly I:C stimulation. Our study is the first detailed survey of the gene expression stability in whole blood and PBMC with or without poly I:C stimulation and should be helpful for investigating the molecular mechanism involved in porcine whole blood and PBMC in response to poly I:C stimulation.

Keywords

References

  1. Andersen, C. L., J. L. Jensen, and T. F. Orntoft. 2004. 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. 64:5245-5250. https://doi.org/10.1158/0008-5472.CAN-04-0496
  2. Brym, P., A. Rusc, and S. Kaminski. 2013. Evaluation of reference genes for qRT-PCR gene expression studies in whole blood samples from healthy and leukemia-virus infected cattle. Vet. Immunol. Immunopathol. 153:302-307. https://doi.org/10.1016/j.vetimm.2013.03.004
  3. Cao, Y., Z. Lu, Y. Li, P. Sun, D. Li, P. Li, X. Bai, Y. Fu, H. Bao, and C. Zhou. 2013. Poly (I: C) combined with multi-epitope protein vaccine completely protects against virulent foot-andmouth disease virus challenge in pigs. Antiviral Res. 97:145-153. https://doi.org/10.1016/j.antiviral.2012.11.009
  4. Caskey, M., F. Lefebvre, A. Filali-Mouhim, M. J. Cameron, J. P. Goulet, E. K. Haddad, G. Breton, C. Trumpfheller, S. Pollak, I. Shimeliovich, A. Duque-Alarcon, L. Pan, A. Nelkenbaum, A. M. Salazar, S. J. Schlesinger, R. M. Steinman, and R. P. Sekaly. 2011. Synthetic double-stranded RNA induces innate immune responses similar to a live viral vaccine in humans. J. Exp. Med. 208:2357-2366. https://doi.org/10.1084/jem.20111171
  5. Cinar, M. U., M. A. Islam, M. Proll, H. Kocamis, E. Tholen, D. Tesfaye, C. Looft, K. Schellander, and M. J. Uddin. 2013. Evaluation of suitable reference genes for gene expression studies in porcine PBMCs in response to LPS and LTA. BMC Res. Notes 6:56. https://doi.org/10.1186/1756-0500-6-56
  6. Cinar, M. U., M. A. Islam, M. J. Uddin, E. Tholen, D. Tesfaye, C. Looft, and K. Schellander. 2012. Evaluation of suitable reference genes for gene expression studies in porcine alveolar macrophages in response to LPS and LTA. BMC Res. Notes 5:107. https://doi.org/10.1186/1756-0500-5-107
  7. Facci, M. R., G. Auray, F. Meurens, R. Buchanan, J. van Kessel, and V. Gerdts. 2011. Stability of expression of reference genes in porcine peripheral blood mononuclear and dendritic cells. Vet. Immunol. Immunopathol. 141:11-15. https://doi.org/10.1016/j.vetimm.2011.01.005
  8. Gao, Y., L. Flori, J. Lecardonnel, D. Esquerre , Z. L. Hu, A. Teillaud, G. Lemonnier, F. Lefevre, I. Oswald, and C. Rogel-Gaillard. 2010. Transcriptome analysis of porcine PBMCs after in vitro stimulation by LPS or PMA/ionomycin using an expression array targeting the pig immune response. BMC Genomics 11:292. https://doi.org/10.1186/1471-2164-11-292
  9. Hellemans, J., G. Mortier, A. De Paepe, F. Speleman, and J. Vandesompele. 2007. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 8:R19. https://doi.org/10.1186/gb-2007-8-2-r19
  10. Huggett, J., K. Dheda, S. Bustin, and A. Zumla. 2005. Real-time RT-PCR normalisation; strategies and considerations. Genes Immun. 6:279-284. https://doi.org/10.1038/sj.gene.6364190
  11. Martino, A., M. Cabiati, M. Campan, T. Prescimone, D. Minocci, C. Caselli, A. M. Rossi, D. Giannessi, and S. Del Ry. 2011. Selection of reference genes for normalization of real-time PCR data in minipig heart failure model and evaluation of TNF-$\alpha$ mRNA expression. J. Biotechnol. 153:92-99. https://doi.org/10.1016/j.jbiotec.2011.04.002
  12. McCartney, S., W. Vermi, S. Gilfillan, M. Cella, T. L. Murphy, R. D. Schreiber, K. M. Murphy, and M. Colonna. 2009. Distinct and complementary functions of MDA5 and TLR3 in poly (I: C)-mediated activation of mouse NK cells. J. Exp. Med. 206:2967-2976. https://doi.org/10.1084/jem.20091181
  13. Meurens, F., A. Summerfield, H. Nauwynck, L. Saif, and V. Gerdts. 2012. The pig: a model for human infectious diseases. Trends Microbiol. 20:50-57. https://doi.org/10.1016/j.tim.2011.11.002
  14. Nygard, A. B., C. B. Jorgensen, S. Cirera, and M. Fredholm. 2007. Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR. BMC Mol. Biol. 8:67. https://doi.org/10.1186/1471-2199-8-67
  15. Pfaffl, M. W., A. Tichopad, C. Prgomet, and T. P. Neuvians. 2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-Excel-based tool using pair-wise correlations. Biotechnol. Lett. 26:509-515. https://doi.org/10.1023/B:BILE.0000019559.84305.47
  16. Radonic, A., S. Thulke, H.-G. Bae, M. A. Muller, W. Siegert, and A. Nitsche. 2005. Reference gene selection for quantitative real-time PCR analysis in virus infected cells: SARS corona virus, Yellow fever virus, Human Herpesvirus-6, Camelpox virus and Cytomegalovirus infections. Virol. J. 2:7. https://doi.org/10.1186/1743-422X-2-7
  17. Spalenza, V., F. Girolami, C. Bevilacqua, F. Riondato, R. Rasero, C. Nebbia, P. Sacchi, and P. Martin. 2011. Identification of internal control genes for quantitative expression analysis by real-time PCR in bovine peripheral lymphocytes. Vet. J. 189:278-283. https://doi.org/10.1016/j.tvjl.2010.11.017
  18. Uddin, M., M. Cinar, D. Tesfaye, C. Looft, E. Tholen, and K. Schellander. 2011. Age-related changes in relative expression stability of commonly used housekeeping genes in selected porcine tissues. BMC Res. Notes 4:441. https://doi.org/10.1186/1756-0500-4-441
  19. Uddin, M. J., P. K. Nuro-Gyina, M. A. Islam, D. Tesfaye, E. Tholen, C. Looft, K. Schellander, and M. U. Cinar. 2012. Expression dynamics of Toll-like receptors mRNA and cytokines in porcine peripheral blood mononuclear cells stimulated by bacterial lipopolysaccharide. Vet. Immunol. Immunopathol. 147:211-222. https://doi.org/10.1016/j.vetimm.2012.04.020
  20. Vandesompele, J., K. De Preter, F. Pattyn, B. Poppe, N. Van Roy, A. De Paepe, and F. Speleman. 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3:research0034- research0034.11.
  21. Xiang-Hong, J., Y. Yan-Hong, X. Han-Jin, A. Li-long, X. Ying-Mei, J. Pei-Rong, and L. Ming. 2011. Selection of reference genes for gene expression studies in PBMC from Bama miniature pig under heat stress. Vet. Immunol. Immunopathol. 144:160-166. https://doi.org/10.1016/j.vetimm.2011.07.004
  22. Zhang, J., Z. Tang, N. Wang, L. Long, and K. Li. 2012. Evaluating a set of reference genes for expression normalization in multiple tissues and skeletal muscle at different development stages in pigs using quantitative real-time polymerase chain reaction. DNA Cell Biol. 31:106-113. https://doi.org/10.1089/dna.2011.1249

Cited by

  1. Transcriptomic landscape for lymphocyte count variation in poly I:C-induced porcine peripheral blood vol.47, pp.1, 2015, https://doi.org/10.1111/age.12379
  2. Validation of Reference Genes for Quantitative Real-Time PCR during Bicolor Tepal Development in Asiatic Hybrid Lilies (Lilium spp.) vol.8, pp.1664-462X, 2017, https://doi.org/10.3389/fpls.2017.00669
  3. MicroRNA Transcriptome of Poly I:C-Stimulated Peripheral Blood Mononuclear Cells Reveals Evidence for MicroRNAs in Regulating Host Response to RNA Viruses in Pigs vol.17, pp.10, 2016, https://doi.org/10.3390/ijms17101601
  4. Transcriptional Profiling of Leucocyte Count Variation from Porcine Peripheral Blood Reveals Differential Gene Expression vol.2018, pp.2314-6141, 2018, https://doi.org/10.1155/2018/1496536
  5. Differential expression of long non-coding RNA and mRNA in children with Henoch-Schönlein purpura nephritis vol.17, pp.1, 2014, https://doi.org/10.3892/etm.2018.7038
  6. Novel Porcine Retina Cultivation Techniques Provide Improved Photoreceptor Preservation vol.14, pp.None, 2014, https://doi.org/10.3389/fnins.2020.556700
  7. Weaning Induces Stress-Dependent DNA Methylation and Transcriptional Changes in Piglet PBMCs vol.12, pp.None, 2021, https://doi.org/10.3389/fgene.2021.633564
  8. Serial blood cytokine and chemokine mRNA and microRNA over 48 h are insult specific in a piglet model of inflammation-sensitized hypoxia-ischaemia vol.89, pp.3, 2014, https://doi.org/10.1038/s41390-020-0986-3