title: Evaluation of suitable reference genes for gene expression studies in porcine PBMCs in response to LPS and LTA.
abstract: BACKGROUND
  As an in vitro model porcine peripheral blood mononuclear cells (PBMCs) is frequently used as for immunogenetic research with the stimulation of bacterial antigens. To investigate the immunocompetence of PBMCs for recognition of Gram-positive and Gram-negative bacteria and in order to dissect the pathogenesis of diseases, gene expression assay is most commonly used. The gene expressions are required to normalize for reference genes which have tremendous effect on the results of expression study. The reference genes should be stably expressed between different cells under a variety of experimental conditions, but recent influx of data showed that expression stability of reference genes are varied under different experimental conditions. But data regarding the expression stability of reference genes in porcine PBMCs are limited. Therefore, this study was aimed to know whether the expression stability of commonly used reference genes in PBMCs is affected by various bacterial antigens under different experimental conditions in pigs.
  
  
  RESULTS
  The mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined by RT-qPCR in PBMCs that were stimulated by LPS and LTA in vitro as well as cells un-stimulated control and non-cultured were also consider for this experiment. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (P < 0.05). geNorm software revealed that in case of irrespective of stimulation (without considering the type of stimulation), RPL4, PPIA and B2M were the most stable reference genes in PBMCs; in case of the control group, PPIA, BLM and GAPDH were the most stable reference genes. PPIA, B2M and RPL4 were the most stable reference genes in LPS stimulated PBMCs; and YWHAZ, RPL4 and PPIA were the most stably expressed reference genes in the case of LTA stimulated PBMCs. When LPS was used combined with LTA for the stimulation, YWHAZ, B2M and SDHA remained the most stable genes. PPIA, BLM and GAPDH were found to be most stably expressed reference genes when PBMCs were not cultured. NormFinder revealed different sets of stably expressed reference genes in PBMCs under different experimental conditions. Moreover, geNorm software suggested that the geometric mean of the three most stable genes would be the suitable combination for accurate normalization of gene expression study.
  
  
  CONCLUSIONS
  There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms (geNorm and NormFinder). In conclusion, the geometric mean of the RPL4, B2M and PPIA seemed to be the most appropriate combination of reference genes for accurate normalization of gene expression data in porcine PBMCs without knowing the type of bacterial pathogenic status of the animals and in the case of mixed infection with Gram-negative and Gram-positive bacteria. In case of PBMCs without any stimulation, PPIA, BLM and GAPDH could be suggested as suitable reference genes.
authors: Institute of Animal Sciences, Unit of Animal Breeding and Husbandry, University of Bonn, Endenicher Allee 15, 53115 Bonn, Germany.
authors: Mehmet Ulas
authors: MU
authors: Cinar
authors: Mohammad Ariful
authors: MA
authors: Islam
authors: Maren
authors: M
authors: Pröll
authors: Hakan
authors: H
authors: Kocamis
authors: Ernst
authors: E
authors: Tholen
authors: Dawit
authors: D
authors: Tesfaye
authors: Christian
authors: C
authors: Looft
authors: Karl
authors: K
authors: Schellander
authors: Muhammad Jasim
authors: MJ
authors: Uddin
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D015870:Gene Expression; D066298:In Vitro Techniques; D008070:Lipopolysaccharides / Q000494:pharmacology*; D009000:Monocytes / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D013552:Swine; D013682:Teichoic Acids / Q000494:pharmacology*
chemical_list: D017931:DNA Primers; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D013682:Teichoic Acids; C009900:lipoteichoic acid
references: BMC Mol Biol. 2009 Jul 20;10:71
references: 19619301
references: Appl Environ Microbiol. 2005 Jun;71(6):2949-54
references: 15932989
references: BMC Mol Biol. 2008 Jul 31;9:69
references: 18671841
references: Biotechniques. 2004 Jul;37(1):112-4, 116, 118-9
references: 15283208
references: Thorax. 2002 Sep;57(9):765-70
references: 12200519
references: Methods Mol Biol. 2009;517:3-14
references: 19378036
references: BMC Biotechnol. 2006 Oct 09;6:41
references: 17026777
references: Nature. 2010 Jan 28;463(7280):485-92
references: 20110992
references: BMC Genomics. 2010 May 11;11:292
references: 20459780
references: Cancer Res. 2004 Aug 1;64(15):5245-50
references: 15289330
references: Genome Biol. 2002 Jun 18;3(7):RESEARCH0034
references: 12184808
references: BMC Immunol. 2010 May 04;11:21
references: 20441576
references: Vet Immunol Immunopathol. 2011 May 15;141(1-2):11-5
references: 21354629
references: BMC Mol Biol. 2009 Jul 02;10:66
references: 19573231
references: BMC Mol Biol. 2011 Jan 28;12:5
references: 21272375
references: Mol Biol Rep. 2012 Aug;39(8):7965-72
references: 22544575
references: J Anim Breed Genet. 2011 Aug;128(4):319-25
references: 21749479
references: BMC Res Notes. 2011 Oct 24;4:440
references: 22023793
references: J Immunol Methods. 2000 Jan 13;233(1-2):83-93
references: 10648859
references: Anal Biochem. 2009 Apr 15;387(2):238-42
references: 19454243
references: BMC Res Notes. 2012 Feb 18;5:107
references: 22340302
references: Biochem Biophys Res Commun. 2011 Apr 22;407(4):758-63
references: 21439935
references: BMC Mol Biol. 2010 Feb 15;11:15
references: 20156357
references: Genome Biol. 2007;8(2):R19
references: 17291332
references: Vet Immunol Immunopathol. 2012 Feb 15;145(3-4):611-7
references: 22297149
references: BMC Mol Biol. 2007 Aug 15;8:67
references: 17697375
references: BMC Res Notes. 2011 Oct 24;4:441
references: 22023805
references: Meat Sci. 2011 Mar;87(3):191-5
references: 21041039
references: Vet Immunol Immunopathol. 2012 Mar 15;146(1):62-73
references: 22365308
references: Vet Immunol Immunopathol. 2001 May 10;79(1-2):41-52
references: 11356249
references: Dev Comp Immunol. 2010 May;34(5):562-71
references: 20045027
references: Stem Cell Res Ther. 2010 Mar 30;1(1):7
references: 20504288
references: PLoS One. 2011;6(5):e19556
references: 21655321
references: Vet Immunol Immunopathol. 2011 Nov 15;144(1-2):160-6
references: 21820186
references: J Biomol Tech. 2006 Apr;17(2):157-62
references: 16741243
references: Eur Respir J. 2006 Feb;27(2):300-6
references: 16452584
references: Biotechnol Lett. 2004 Mar;26(6):509-15
references: 15127793
references: J Biochem Biophys Methods. 2000 Nov 20;46(1-2):69-81
references: 11086195
references: Genes Immun. 2005 Jun;6(4):279-84
references: 15815687
references: J Gen Virol. 2003 Oct;84(Pt 10):2755-2760
references: 13679610
references: BMC Res Notes. 2011 Oct 14;4:410
references: 21996334
references: Vet Immunol Immunopathol. 2007 Jul 15;118(1-2):140-6
references: 17544155
references: Vet Immunol Immunopathol. 2012 Jun 30;147(3-4):211-22
references: 22578850
references: Biochem Biophys Res Commun. 2004 Jan 23;313(4):856-62
references: 14706621
references: J Mol Endocrinol. 2002 Aug;29(1):23-39
references: 12200227
references: Vet Immunol Immunopathol. 2011 Feb 15;139(2-4):296-302
references: 21075456
references: J Biomol Tech. 2004 Sep;15(3):155-66
references: 15331581
references: Vet Immunol Immunopathol. 2008 Apr 15;122(3-4):312-7
references: 18272235
references: J Anim Sci. 2011 Oct;89(10):3016-29
references: 21622880