FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / BMC Mol Biol / Evaluation of suitable reference genes for gene expression s...
BMC molecular biology2011; 12; 5; doi: 10.1186/1471-2199-12-5

Evaluation of suitable reference genes for gene expression studies in bronchoalveolar lavage cells from horses with inflammatory airway disease.

Abstract: The stability of reference genes has a tremendous effect on the results of relative quantification of genes expression by quantitative polymerase chain reaction. Equine Inflammatory Airway Disease (IAD) is a common condition often treated with corticosteroids. The diagnosis of IAD is based on clinical signs and bronchoalveolar lavage (BAL) fluid cytology. The aim of this study was to identify reference genes with the most stable mRNA expression in the BAL cells of horses with IAD irrespective of corticosteroids treatment. Results: The expression stability of seven candidate reference genes (B2M, HPRT, GAPDH, ACTB, UBB, RPL32, SDHA) was determined by qRT-PCR in BAL samples taken pre- and post- treatment with dexamethasone and fluticasone propionate for two weeks in 7 horses with IAD. Primers' efficiencies were calculated using LinRegPCR. NormFinder, GeNorm and qBasePlus softwares were used to rank the genes according to their stability. GeNorm was also used to determine both the ideal number and the best combination of reference genes. GAPDH was found to be the most stably expressed gene with the three softwares. GeNorm ranked B2M as the least stable gene. Based on the pair-wise variation cut-off value determined with GeNorm, the number of genes required for optimal normalization was four and included GAPDH, SDHA, HPRT and RPL32. Conclusions: The geometric mean of GAPDH, HPRT, SDHA and RPL32 is recommended for accurate normalization of quantitative PCR data in BAL cells of horses with IAD treated with corticosteroids. If only one reference gene can be used, then GAPDH is recommended.
Get Full Text
Publication Date: 2011-01-28 PubMed ID: 21272375PubMed Central: PMC3039571DOI: 10.1186/1471-2199-12-5Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article
  • Research Support
  • Non-U.S. Gov't
  • Validation Study

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research paper focuses on identifying the most stable reference genes for gene expression studies in horses with Inflammatory Airway Disease (IAD) under corticosteroids treatment. The study finds the geometric mean of GAPDH, HPRT, SDHA and RPL32 is recommended for accurate normalization, and if only one reference gene can be used, GAPDH is the most reliable.

Study Objective

  • The research’s aim was to find reference genes with the greatest stability in mRNA expression in bronchoalveolar lavage (BAL) cells from horses with Inflammatory Airway Disease (IAD), irrespective of corticosteroids treatment.

Methodology

  • The study evaluated the stability of seven proposed reference genes (B2M, HPRT, GAPDH, ACTB, UBB, RPL32, SDHA) through quantitative reverse transcription polymerase chain reaction (qRT-PCR) in BAL samples taken pre- and post- dexamethasone and fluticasone propionate treatment in 7 horses with IAD.
  • The primers for this experiment were calculated via LinRegPCR while NormFinder, GeNorm and qBasePlus softwares aided in ranking the genes based on their stability. GeNorm software was further used to identify the ideal number and combination of reference genes.

Findings

  • The most stably expressed gene across all the software used for the study was GAPDH, B2M was found to be the least stable gene.
  • Based on the pair-wise variation cut-off value determined by GeNorm, four genes namely GAPDH, SDHA, HPRT and RPL32 were required for the optimal normalization of results.

Conclusion

  • For accurate normalization of quantitative PCR data in BAL cells of horses with IAD treated with corticosteroids, the geometric mean of selected genes GAPDH, HPRT, SDHA and RPL32 was recommended.
  • In cases where only one reference gene can be used, the study recommends GAPDH as the preferred option.

Cite This Article

APA
Beekman L, Tohver T, Dardari R, Léguillette R. (2011). Evaluation of suitable reference genes for gene expression studies in bronchoalveolar lavage cells from horses with inflammatory airway disease. BMC Mol Biol, 12, 5. https://doi.org/10.1186/1471-2199-12-5

Publication

BMC molecular biology
ISSN: 1471-2199
NlmUniqueID: 100966983
Country: England
Language: English
Volume: 12
Pages: 5

Researcher Affiliations

Beekman, Laura
  • Departement of Veterinary Clinical and Diagnostic Sciences, Faculty of Veterinary Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada.
Tohver, Triin
    Dardari, Rkia
      Léguillette, Renaud

        MeSH Terms

        • Animals
        • Bronchoalveolar Lavage / veterinary
        • Bronchoalveolar Lavage Fluid / cytology
        • Female
        • Gene Expression Profiling
        • Horse Diseases / diagnosis
        • Horse Diseases / genetics
        • Horses
        • Inflammation / diagnosis
        • Inflammation / veterinary
        • Lung Diseases / genetics
        • Lung Diseases / veterinary
        • Male
        • Reference Standards
        • Reverse Transcriptase Polymerase Chain Reaction

        References

        This article includes 43 references
        1. Hoffman A, Robinson NE, Wade JF. Proceedings of a workshop on Inflammatory Airway Disease: Defining the syndrome. Havemeyer Foundation Boston USA; 2002.
        2. Couëtil LL, Hoffman AM, Hodgson J, Buechner-Maxwell V, Viel L, Wood JL, Lavoie JP. Inflammatory airway disease of horses.. J Vet Intern Med 2007 Mar-Apr;21(2):356-61.
          doi: 10.1111/j.1939-1676.2007.tb02975.xpubmed: 17427403google scholar: lookup
        3. Allen KJ, Tremaine WH, Franklin SH. Prevalence of inflammatory airway disease in national hunt horses referred for investigation of poor athletic performance.. Equine Vet J Suppl 2006 Aug;(36):529-34.
          pubmed: 17402478doi: 10.1111/j.2042-3306.2006.tb05599.xgoogle scholar: lookup
        4. Richard EA, Fortier GD, Lekeux PM, Van Erck E. Laboratory findings in respiratory fluids of the poorly-performing horse.. Vet J 2010 Aug;185(2):115-22.
          doi: 10.1016/j.tvjl.2009.05.003pubmed: 19481964google scholar: lookup
        5. Wasko AJ, Barkema HW, Nicol J, Fernandez N, Logie N, Léguillette R. Evaluation of a risk-screening questionnaire to detect equine lung inflammation: results of a large field study.. Equine Vet J 2011 Mar;43(2):145-52.
          pubmed: 21592207doi: 10.1111/j.2042-3306.2010.00150.xgoogle scholar: lookup
        6. Giguère S, Viel L, Lee E, MacKay RJ, Hernandez J, Franchini M. Cytokine induction in pulmonary airways of horses with heaves and effect of therapy with inhaled fluticasone propionate.. Vet Immunol Immunopathol 2002 Mar;85(3-4):147-58.
          doi: 10.1016/S0165-2427(01)00420-2pubmed: 11943316google scholar: lookup
        7. DeLuca L, Erb HN, Young JC, Perkins GA, Ainsworth DM. The effect of adding oral dexamethasone to feed alterations on the airway cell inflammatory gene expression in stabled horses affected with recurrent airway obstruction.. J Vet Intern Med 2008 Mar-Apr;22(2):427-35.
          doi: 10.1111/j.1939-1676.2008.0055.xpubmed: 18346142google scholar: lookup
        8. Arya M, Shergill IS, Williamson M, Gommersall L, Arya N, Patel HR. Basic principles of real-time quantitative PCR.. Expert Rev Mol Diagn 2005 Mar;5(2):209-19.
          doi: 10.1586/14737159.5.2.209pubmed: 15833050google scholar: lookup
        9. Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real-time RT-PCR.. Nat Protoc 2006;1(3):1559-82.
          doi: 10.1038/nprot.2006.236pubmed: 17406449google scholar: lookup
        10. Huggett J, Dheda K, Bustin S, Zumla A. Real-time RT-PCR normalisation; strategies and considerations.. Genes Immun 2005 Jun;6(4):279-84.
          doi: 10.1038/sj.gene.6364190pubmed: 15815687google scholar: lookup
        11. Schmittgen TD, Zakrajsek BA. Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR.. J Biochem Biophys Methods 2000 Nov 20;46(1-2):69-81.
          doi: 10.1016/S0165-022X(00)00129-9pubmed: 11086195google scholar: lookup
        12. Waxman S, Wurmbach E. De-regulation of common housekeeping genes in hepatocellular carcinoma.. BMC Genomics 2007 Jul 18;8:243.
          doi: 10.1186/1471-2164-8-243pmc: PMC1937003pubmed: 17640361google scholar: lookup
        13. Jung M, Ramankulov A, Roigas J, Johannsen M, Ringsdorf M, Kristiansen G, Jung K. In search of suitable reference genes for gene expression studies of human renal cell carcinoma by real-time PCR.. BMC Mol Biol 2007 Jun 8;8:47.
          doi: 10.1186/1471-2199-8-47pmc: PMC1913536pubmed: 17559644google scholar: lookup
        14. Selvey S, Thompson EW, Matthaei K, Lea RA, Irving MG, Griffiths LR. Beta-actin--an unsuitable internal control for RT-PCR.. Mol Cell Probes 2001 Oct;15(5):307-11.
          doi: 10.1006/mcpr.2001.0376pubmed: 11735303google scholar: lookup
        15. Barber RD, Harmer DW, Coleman RA, Clark BJ. GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues.. Physiol Genomics 2005 May 11;21(3):389-95.
          doi: 10.1152/physiolgenomics.00025.2005pubmed: 15769908google scholar: lookup
        16. 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 Jun 18;3(7):RESEARCH0034.
          doi: 10.1186/gb-2002-3-7-research0034pmc: PMC126239pubmed: 12184808google scholar: lookup
        17. Bogaert L, Van Poucke M, De Baere C, Peelman L, Gasthuys F, Martens A. Selection of a set of reliable reference genes for quantitative real-time PCR in normal equine skin and in equine sarcoids.. BMC Biotechnol 2006 Apr 27;6:24.
          doi: 10.1186/1472-6750-6-24pmc: PMC1484482pubmed: 16643647google scholar: lookup
        18. Zhang YW, Davis EG, Bai J. Determination of internal control for gene expression studies in equine tissues and cell culture using quantitative RT-PCR.. Vet Immunol Immunopathol 2009 Jul 15;130(1-2):114-9.
          doi: 10.1016/j.vetimm.2009.01.012pubmed: 19269038google scholar: lookup
        19. Cappelli K, Felicetti M, Capomaccio S, Spinsanti G, Silvestrelli M, Supplizi AV. Exercise induced stress in horses: selection of the most stable reference genes for quantitative RT-PCR normalization.. BMC Mol Biol 2008 May 19;9:49.
          doi: 10.1186/1471-2199-9-49pmc: PMC2412902pubmed: 18489742google scholar: lookup
        20. Figueiredo MD, Salter CE, Andrietti AL, Vandenplas ML, Hurley DJ, Moore JN. Validation of a reliable set of primer pairs for measuring gene expression by real-time quantitative RT-PCR in equine leukocytes.. Vet Immunol Immunopathol 2009 Sep 15;131(1-2):65-72.
          doi: 10.1016/j.vetimm.2009.03.013pubmed: 19376596google scholar: lookup
        21. Kriegova E, Arakelyan A, Fillerova R, Zatloukal J, Mrazek F, Navratilova Z, Kolek V, du Bois RM, Petrek M. PSMB2 and RPL32 are suitable denominators to normalize gene expression profiles in bronchoalveolar cells.. BMC Mol Biol 2008 Jul 31;9:69.
          doi: 10.1186/1471-2199-9-69pmc: PMC2529339pubmed: 18671841google scholar: lookup
        22. Glare EM, Divjak M, Bailey MJ, Walters EH. beta-Actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels.. Thorax 2002 Sep;57(9):765-70.
          doi: 10.1136/thorax.57.9.765pmc: PMC1746418pubmed: 12200519google scholar: lookup
        23. Fu LY, Jia HL, Dong QZ, Wu JC, Zhao Y, Zhou HJ, Ren N, Ye QH, Qin LX. Suitable reference genes for real-time PCR in human HBV-related hepatocellular carcinoma with different clinical prognoses.. BMC Cancer 2009 Feb 6;9:49.
          doi: 10.1186/1471-2407-9-49pmc: PMC2644316pubmed: 19200351google scholar: lookup
        24. 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 Sep 1;344(1):141-3.
          doi: 10.1016/j.ab.2005.05.022pubmed: 16054107google scholar: lookup
        25. Tatsumi K, Ohashi K, Taminishi S, Okano T, Yoshioka A, Shima M. Reference gene selection for real-time RT-PCR in regenerating mouse livers.. Biochem Biophys Res Commun 2008 Sep 12;374(1):106-10.
          doi: 10.1016/j.bbrc.2008.06.103pubmed: 18602371google scholar: lookup
        26. Taylor SE, Vaughan-Thomas A, Clements DN, Pinchbeck G, Macrory LC, Smith RK, Clegg PD. Gene expression markers of tendon fibroblasts in normal and diseased tissue compared to monolayer and three dimensional culture systems.. BMC Musculoskelet Disord 2009 Feb 26;10:27.
          doi: 10.1186/1471-2474-10-27pmc: PMC2651848pubmed: 19245707google scholar: lookup
        27. von Rechenberg B, Leutenegger C, Zlinsky K, McIlwraith CW, Akens MK, Auer JA. Upregulation of mRNA of interleukin-1 and -6 in subchondral cystic lesions of four horses.. Equine Vet J 2001 Mar;33(2):143-9.
          doi: 10.1111/j.2042-3306.2001.tb00592.xpubmed: 11266063google scholar: lookup
        28. Ishii T, Wallace AM, Zhang X, Gosselink J, Abboud RT, English JC, Paré PD, Sandford AJ. Stability of housekeeping genes in alveolar macrophages from COPD patients.. Eur Respir J 2006 Feb;27(2):300-6.
          doi: 10.1183/09031936.06.00090405pubmed: 16452584google scholar: lookup
        29. Liu DW, Chen ST, Liu HP. Choice of endogenous control for gene expression in nonsmall cell lung cancer.. Eur Respir J 2005 Dec;26(6):1002-8.
          doi: 10.1183/09031936.05.00050205pubmed: 16319328google scholar: lookup
        30. Willems E, Mateizel I, Kemp C, Cauffman G, Sermon K, Leyns L. Selection of reference genes in mouse embryos and in differentiating human and mouse ES cells.. Int J Dev Biol 2006;50(7):627-35.
          doi: 10.1387/ijdb.052130ewpubmed: 16892176google scholar: lookup
        31. Andersen CL, Jensen JL, Ørntoft 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 Aug 1;64(15):5245-50.
          doi: 10.1158/0008-5472.CAN-04-0496pubmed: 15289330google scholar: lookup
        32. O'Connor AM, Sargeant JM, Gardner IA, Dickson JS, Torrence ME, Dewey CE, Dohoo IR, Evans RB, Gray JT, Greiner M, Keefe G, Lefebvre SL, Morley PS, Ramirez A, Sischo W, Smith DR, Snedeker K, Sofos J, Ward MP, Wills R. The REFLECT statement: methods and processes of creating reporting guidelines for randomized controlled trials for livestock and food safety.. J Vet Intern Med 2010 Jan-Feb;24(1):57-64.
          pubmed: 20002546doi: 10.1111/j.1939-1676.2009.0441.xgoogle scholar: lookup
        33. Ainsworth DM, Matychak M, Reyner CL, Erb HN, Young JC. Effects of in vitro exposure to hay dust on the gene expression of chemokines and cell-surface receptors in primary bronchial epithelial cell cultures established from horses with chronic recurrent airway obstruction.. Am J Vet Res 2009 Mar;70(3):365-72.
          doi: 10.2460/ajvr.70.3.365pubmed: 19254149google scholar: lookup
        34. Reid CJ, Thompson L, Donnelly S, Deaton CM, Marlin DJ, Carrington SD, Irwin JA. The activity and expression of chitinase in the equine lung and its activity in normal horses and animals with recurrent airway obstruction.. Res Vet Sci 2009 Aug;87(1):20-5.
          doi: 10.1016/j.rvsc.2008.11.002pubmed: 19103451google scholar: lookup
        35. Perkins GA, Viel L, Wagner B, Hoffman A, Erb HN, Ainsworth DM. Histamine bronchoprovocation does not affect bronchoalveolar lavage fluid cytology, gene expression and protein concentrations of IL-4, IL-8 and IFN-gamma.. Vet Immunol Immunopathol 2008 Dec 15;126(3-4):230-5.
          doi: 10.1016/j.vetimm.2008.07.006pubmed: 18829118google scholar: lookup
        36. Riihimäki M, Raine A, Pourazar J, Sandström T, Art T, Lekeux P, Couëtil L, Pringle J. Epithelial expression of mRNA and protein for IL-6, IL-10 and TNF-alpha in endobronchial biopsies in horses with recurrent airway obstruction.. BMC Vet Res 2008 Feb 23;4:8.
          doi: 10.1186/1746-6148-4-8pmc: PMC2294109pubmed: 18294392google scholar: lookup
        37. Laan TT, Bull S, Pirie R, Fink-Gremmels J. The role of alveolar macrophages in the pathogenesis of recurrent airway obstruction in horses.. J Vet Intern Med 2006 Jan-Feb;20(1):167-74.
          doi: 10.1111/j.1939-1676.2006.tb02837.xpubmed: 16496937google scholar: lookup
        38. Giguère S, Prescott JF. Quantitation of equine cytokine mRNA expression by reverse transcription-competitive polymerase chain reaction.. Vet Immunol Immunopathol 1999 Jan 4;67(1):1-15.
          doi: 10.1016/S0165-2427(98)00212-8pubmed: 9950350google scholar: lookup
        39. Primer3. http://frodo.wi.mit.edu/primer3/
        40. Browser EG. http://www.ensembl.org
        41. Zuker M. Mfold web server for nucleic acid folding and hybridization prediction.. Nucleic Acids Res 2003 Jul 1;31(13):3406-15.
          doi: 10.1093/nar/gkg595pmc: PMC169194pubmed: 12824337google scholar: lookup
        42. Ruijter JM, Ramakers C, Hoogaars WM, Karlen Y, Bakker O, van den Hoff MJ, Moorman AF. Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data.. Nucleic Acids Res 2009 Apr;37(6):e45.
          doi: 10.1093/nar/gkp045pmc: PMC2665230pubmed: 19237396google scholar: lookup
        43. Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data.. Genome Biol 2007;8(2):R19.
          doi: 10.1186/gb-2007-8-2-r19pmc: PMC1852402pubmed: 17291332google scholar: lookup

        Citations

        This article has been cited 34 times.
        1. Woodrow JS, Hines M, Sommardahl C, Flatland B, Lo Y, Wang Z, Sheats MK, Lennon EM. Initial investigation of molecular phenotypes of airway mast cells and cytokine profiles in equine asthma. Front Vet Sci 2022;9:997139.
          doi: 10.3389/fvets.2022.997139pubmed: 36713876google scholar: lookup
        2. Adamič N, Prpar Mihevc S, Blagus R, Kramarič P, Krapež U, Majdič G, Viel L, Hoffman AM, Bienzle D, Vengust M. Effect of intrabronchial administration of autologous adipose-derived mesenchymal stem cells on severe equine asthma. Stem Cell Res Ther 2022 Jan 21;13(1):23.
          doi: 10.1186/s13287-022-02704-7pubmed: 35063028google scholar: lookup
        3. Karagianni AE, Kurian D, Cillán-Garcia E, Eaton SL, Wishart TM, Pirie RS. Training associated alterations in equine respiratory immunity using a multiomics comparative approach. Sci Rep 2022 Jan 10;12(1):427.
          doi: 10.1038/s41598-021-04137-3pubmed: 35013475google scholar: lookup
        4. Suntichaikamolkul N, Sangpong L, Schaller H, Sirikantaramas S. Genome-wide identification and expression profiling of durian CYPome related to fruit ripening. PLoS One 2021;16(11):e0260665.
          doi: 10.1371/journal.pone.0260665pubmed: 34847184google scholar: lookup
        5. Xie CD, Wang B, Shen ZJ, Yao WY, Ao H, Li B, Pei Y, Zhou R. Validation of the Reference Genes for the Gene Expression Studies in Different Cell Lines of Pig. Biomed Res Int 2021;2021:5364190.
          doi: 10.1155/2021/5364190pubmed: 34458368google scholar: lookup
        6. Karagianni AE, Eaton SL, Kurian D, Cillán-Garcia E, Twynam-Perkins J, Raper A, Wishart TM, Pirie RS. Application across species of a one health approach to liquid sample handling for respiratory based -omics analysis. Sci Rep 2021 Jul 12;11(1):14292.
          doi: 10.1038/s41598-021-93839-9pubmed: 34253818google scholar: lookup
        7. Torres R, Hurtado C, Pérez-Macchi S, Bittencourt P, Freschi C, de Mello VVC, Machado RZ, André MR, Müller A. Occurrence and Genetic Diversity of Babesia caballi and Theileria equi in Chilean Thoroughbred Racing Horses. Pathogens 2021 Jun 7;10(6).
          doi: 10.3390/pathogens10060714pubmed: 34200433google scholar: lookup
        8. Mainguy-Seers S, Lavoie JP. Glucocorticoid treatment in horses with asthma: A narrative review. J Vet Intern Med 2021 Jul;35(4):2045-2057.
          doi: 10.1111/jvim.16189pubmed: 34085342google scholar: lookup
        9. Davis KU, Sheats MK. Differential gene expression and Ingenuity Pathway Analysis of bronchoalveolar lavage cells from horses with mild/moderate neutrophilic or mastocytic inflammation on BAL cytology. Vet Immunol Immunopathol 2021 Apr;234:110195.
          doi: 10.1016/j.vetimm.2021.110195pubmed: 33588285google scholar: lookup
        10. Hsu D, Kim A, Ganz T, Suh JD, Wang M, Wu TJ, Lee JT. Expression of Iron-Regulatory Hormone Hepcidin and Iron Transporters Ferroportin and ZIP8 in Patients With and Without Chronic Rhinosinusitis. Otolaryngol Head Neck Surg 2020 Dec;163(6):1270-1273.
          doi: 10.1177/0194599820939910pubmed: 32746765google scholar: lookup
        11. Hue E, Orard M, Toquet MP, Depecker M, Couroucé A, Pronost S, Paillot R, Richard EA. Asymmetrical Pulmonary Cytokine Profiles Are Linked to Bronchoalveolar Lavage Fluid Cytology of Horses With Mild Airway Neutrophilia. Front Vet Sci 2020;7:226.
          doi: 10.3389/fvets.2020.00226pubmed: 32391392google scholar: lookup
        12. Bond SL, Hundt J, Léguillette R. Effect of injected dexamethasone on relative cytokine mRNA expression in bronchoalveolar lavage fluid in horses with mild asthma. BMC Vet Res 2019 Nov 6;15(1):397.
          doi: 10.1186/s12917-019-2144-xpubmed: 31694631google scholar: lookup
        13. Cheevarungnapakul K, Khaksar G, Panpetch P, Boonjing P, Sirikantaramas S. Identification and Functional Characterization of Genes Involved in the Biosynthesis of Caffeoylquinic Acids in Sunflower (Helianthus annuus L.). Front Plant Sci 2019;10:968.
          doi: 10.3389/fpls.2019.00968pubmed: 31417585google scholar: lookup
        14. Cartwright JA, Gow AG, Milne E, Drummond D, Smith S, Handel I, Mellanby RJ. Vitamin D Receptor Expression in Dogs. J Vet Intern Med 2018 Mar;32(2):764-774.
          doi: 10.1111/jvim.15052pubmed: 29469965google scholar: lookup
        15. Samiullah S, Roberts J, Wu SB. Reference gene selection for the shell gland of laying hens in response to time-points of eggshell formation and nicarbazin. PLoS One 2017;12(7):e0180432.
          doi: 10.1371/journal.pone.0180432pubmed: 28671969google scholar: lookup
        16. Davidsson P, Broberg M, Kariola T, Sipari N, Pirhonen M, Palva ET. Short oligogalacturonides induce pathogen resistance-associated gene expression in Arabidopsis thaliana. BMC Plant Biol 2017 Jan 19;17(1):19.
          doi: 10.1186/s12870-016-0959-1pubmed: 28103793google scholar: lookup
        17. Survila M, Davidsson PR, Pennanen V, Kariola T, Broberg M, Sipari N, Heino P, Palva ET. Peroxidase-Generated Apoplastic ROS Impair Cuticle Integrity and Contribute to DAMP-Elicited Defenses. Front Plant Sci 2016;7:1945.
          doi: 10.3389/fpls.2016.01945pubmed: 28066496google scholar: lookup
        18. Azarpeykan S, Dittmer KE. Evaluation of housekeeping genes for quantitative gene expression analysis in the equine kidney. J Equine Sci 2016;27(4):165-168.
          doi: 10.1294/jes.27.165pubmed: 27974876google scholar: lookup
        19. Uddin MJ, Suen WW, Bosco-Lauth A, Hartwig AE, Hall RA, Bowen RA, Bielefeldt-Ohmann H. Kinetics of the West Nile virus induced transcripts of selected cytokines and Toll-like receptors in equine peripheral blood mononuclear cells. Vet Res 2016 Jun 7;47(1):61.
          doi: 10.1186/s13567-016-0347-8pubmed: 27267361google scholar: lookup
        20. Scarlet D, Ertl R, Aurich C, Steinborn R. The Orthology Clause in the Next Generation Sequencing Era: Novel Reference Genes Identified by RNA-seq in Humans Improve Normalization of Neonatal Equine Ovary RT-qPCR Data. PLoS One 2015;10(11):e0142122.
          doi: 10.1371/journal.pone.0142122pubmed: 26536597google scholar: lookup
        21. Altmann S, Rebl A, Kühn C, Goldammer T. Identification and de novo sequencing of housekeeping genes appropriate for gene expression analyses in farmed maraena whitefish (Coregonus maraena) during crowding stress. Fish Physiol Biochem 2015 Apr;41(2):397-412.
          doi: 10.1007/s10695-014-9991-ypubmed: 25249196google scholar: lookup
        22. Ji H, Wang J, Liu J, Guo J, Wang Z, Zhang X, Guo L, Yang H. Selection of Reliable Reference Genes for Real-time qRT-PCR Analysis of Zi Geese (Anser anser domestica) Gene Expression. Asian-Australas J Anim Sci 2013 Mar;26(3):423-32.
          doi: 10.5713/ajas.2012.12417pubmed: 25049806google scholar: lookup
        23. Najafpanah MJ, Sadeghi M, Bakhtiarizadeh MR. Reference genes selection for quantitative real-time PCR using RankAggreg method in different tissues of Capra hircus. PLoS One 2013;8(12):e83041.
          doi: 10.1371/journal.pone.0083041pubmed: 24358246google scholar: lookup
        24. Kianianmomeni A, Hallmann A. Validation of reference genes for quantitative gene expression studies in Volvox carteri using real-time RT-PCR. Mol Biol Rep 2013 Dec;40(12):6691-9.
          doi: 10.1007/s11033-013-2784-zpubmed: 24057254google scholar: lookup
        25. Karagianni AE, Kapetanovic R, McGorum BC, Hume DA, Pirie SR. The equine alveolar macrophage: functional and phenotypic comparisons with peritoneal macrophages. Vet Immunol Immunopathol 2013 Oct 1;155(4):219-28.
          doi: 10.1016/j.vetimm.2013.07.003pubmed: 23978307google scholar: lookup
        26. Cinar MU, Islam MA, Pröll M, Kocamis H, Tholen E, Tesfaye D, Looft C, Schellander K, Uddin MJ. Evaluation of suitable reference genes for gene expression studies in porcine PBMCs in response to LPS and LTA. BMC Res Notes 2013 Feb 8;6:56.
          doi: 10.1186/1756-0500-6-56pubmed: 23394600google scholar: lookup
        27. Chooi WH, Zhou R, Yeo SS, Zhang F, Wang DA. Determination and validation of reference gene stability for qPCR analysis in polysaccharide hydrogel-based 3D chondrocytes and mesenchymal stem cell cultural models. Mol Biotechnol 2013 Jun;54(2):623-33.
          doi: 10.1007/s12033-012-9604-xpubmed: 23054629google scholar: lookup
        28. Cinar MU, Islam MA, Uddin MJ, Tholen E, Tesfaye D, Looft C, Schellander K. Evaluation of suitable reference genes for gene expression studies in porcine alveolar macrophages in response to LPS and LTA. BMC Res Notes 2012 Feb 18;5:107.
          doi: 10.1186/1756-0500-5-107pubmed: 22340302google scholar: lookup
        29. Uddin MJ, Cinar MU, Tesfaye D, Looft C, Tholen E, Schellander K. Age-related changes in relative expression stability of commonly used housekeeping genes in selected porcine tissues. BMC Res Notes 2011 Oct 24;4:441.
          doi: 10.1186/1756-0500-4-441pubmed: 22023805google scholar: lookup
        30. Sanz MG, Jellen G, Cody L, Bergsma J, Cha M, Kogan C, Kordas G, Bayly WM, Leguillette R. Use of inhaled ciclesonide for treatment of moderate asthma in Thoroughbred racehorses. J Vet Intern Med 2025 Mar-Apr;39(2):e17267.
          doi: 10.1111/jvim.17267pubmed: 39945569google scholar: lookup
        31. Sharma NK, Singh P, Saha B, Bhardwaj A, Iquebal MA, Pal Y, Nayan V, Jaiswal S, Giri SK, Legha RA, Bhattacharya TK, Kumar D, Rai A. Genome wide landscaping of copy number variations for horse inter-breed variability. Anim Biotechnol 2025 Dec;36(1):2446251.
          doi: 10.1080/10495398.2024.2446251pubmed: 39791493google scholar: lookup
        32. Mazzei M, Sorvillo B, Sgorbini M, Bindi F, Perelli A, Laus F. The Role of Viral Pathogens in Horse Respiratory Diseases: A Cytological and Molecular Approach Using Next-Generation Sequencing. Animals (Basel) 2024 Nov 21;14(23).
          doi: 10.3390/ani14233347pubmed: 39682313google scholar: lookup
        33. Bond S, Léguillette R. A CONSORT-guided, randomized controlled clinical trial of nebulized administration of dexamethasone and saline on lower airway cytokine mRNA expression in horses with moderate asthma. J Vet Intern Med 2024 Mar-Apr;38(2):1214-1223.
          doi: 10.1111/jvim.16983pubmed: 38205666google scholar: lookup
        34. Ayakannu T, Taylor AH, Konje JC. Selection of Endogenous Control Reference Genes for Studies on Type 1 or Type 2 Endometrial Cancer. Sci Rep 2020 May 21;10(1):8468.
          doi: 10.1038/s41598-020-64663-4pubmed: 32439920google scholar: lookup
        Subscribe to our newsletter
        Join 99,001 horse owners like you who receive our email updates on horse health and nutrition.