, Volume 23, Issue 5, pp 625–633 | Cite as

Reference genes for real-time RT-PCR expression studies in an Antarctic Pseudomonas exposed to different temperature conditions

  • César X. García-Laviña
  • Susana Castro-Sowinski
  • Ana RamónEmail author
Method Paper


Psychrophilic and psychrotolerant bacteria from permanently cold environments may be the most abundant extremophiles on Earth and yet little is known on how they cope with temperature stress. Real-time reverse transcription PCR (RT-qPCR) is a powerful technique that could shed light on this matter but it requires pre-validated reference genes for normalization of data to get accurate results. In this study, we assessed the expression stability of eight candidate genes for the psychrotolerant Antarctic isolate Pseudomonas sp. AU10 during exponential growth under 4 °C and 30 °C, and after a cold-shock. Using the software programs BestKeeper and geNorm we validated recA, ftsZ, 16S rRNA, and rpoD as reference genes and we suggested the combination of recA and ftsZ for qPCR data normalization. Our results provide a starting point for gene expression studies in Antarctic Pseudomonas concerning temperature-related physiology and also for the validation of reference genes in other cold-adapted bacterial species.


Reference genes Validation RT-qPCR Pseudomonas Psychrotolerant Cold-shock 



The authors especially thank Prof. Lucia Yim and MSc Mailén Arleo for their support and valuable suggestions during this work. They also thank the Uruguayan Antarctic Institute for the logistic support during the stay in the Antarctic Base Artigas. A. Ramón and S. Castro-Sowinski are members of the National Research System (SNI, Sistema Nacional de Investigadores). This work was partially supported by PEDECIBA (Programa de Desarrollo de las Ciencias Básicas) and CSIC (Comisión Sectorial de Investigación Científica, C335-348). The work of CXGL was supported by ANII (Agencia Nacional de Investigación e Innovación) and CAP (Comisión Académica de Posgrado).

Supplementary material

792_2019_1109_MOESM1_ESM.jpg (1.5 mb)
Supplementary file1 (JPG 1494 kb)


  1. Alqarni B, Colley B, Klebensberger J et al (2016) Expression stability of 13 housekeeping genes during carbon starvation of Pseudomonas aeruginosa. J Microbiol Methods 127:182–187. CrossRefGoogle Scholar
  2. Aranda PS, LaJoie DM, Jorcyk CL (2012) Bleach gel: a simple agarose gel for analyzing RNA quality. Electrophoresis 33:366–369. CrossRefGoogle Scholar
  3. Berger F, Morellet N, Menu F, Potier P (1996) Cold shock and cold acclimation proteins in the psychrotrophic bacterium Arthrobacter globiformis SI55. J Bacteriol 178:2999–3007. CrossRefGoogle Scholar
  4. Brudal E, Winther-Larsen HC, Colquhoun DJ, Duodu S (2013) Evaluation of reference genes for reverse transcription quantitative PCR analyses of fish-pathogenic Francisella strains exposed to different growth conditions. BMC Res Notes 6:76. CrossRefGoogle Scholar
  5. Bustin S (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29:23–39. CrossRefGoogle Scholar
  6. Bustin SA, Benes V, Nolan T, Pfaffl MW (2005) Quantitative real-time RT-PCR—a perspective. J Mol Endocrinol 34:597–601. CrossRefGoogle Scholar
  7. Bustin SA, Benes V, Garson JA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622. CrossRefGoogle Scholar
  8. Carrillo-Casas EM, Hernández-Castro R, Suárez-Güemes F, De La Peña-Moctezuma A (2008) Selection of the internal control gene for real-time quantitative RT-PCR assays in temperature treated Leptospira. Curr Microbiol 56:539–546. CrossRefGoogle Scholar
  9. Chang Q, Amemiya T, Liu J et al (2009) Identification and validation of suitable reference genes for quantitative expression of xylA and xylE genes in Pseudomonas putida mt-2. J Biosci Bioeng 107:210–214. CrossRefGoogle Scholar
  10. Czapski TR, Trun N (2014) Expression of csp genes in E. coli K-12 in defined rich and defined minimal media during normal growth, and after cold-shock. Gene 547:91–97. CrossRefGoogle Scholar
  11. Darrieux M, Ribeiro ML, Vicentini R et al (2018) Selection and validation of reference genes for gene expression studies in Klebsiella pneumoniae using reverse transcription quantitative real-time PCR. Sci Rep 8:1–14. CrossRefGoogle Scholar
  12. De Maayer P, Anderson D, Cary C, Cowan DA (2014) Some like it cold: understanding the survival strategies of psychrophiles. EMBO Rep 15:508–517. CrossRefGoogle Scholar
  13. Dheda K, Huggett JF, Chang JS et al (2005) The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization. Anal Biochem 344:141–143. CrossRefGoogle Scholar
  14. Florindo C, Ferreira R, Borges V et al (2012) Selection of reference genes for real-time expression studies in Streptococcus agalactiae. J Microbiol Methods 90:220–227. CrossRefGoogle Scholar
  15. Galisa PS, da Silva HAP, Macedo AVM et al (2012) Identification and validation of reference genes to study the gene expression in Gluconacetobacter diazotrophicus grown in different carbon sources using RT-qPCR. J Microbiol Methods 91:1–7. CrossRefGoogle Scholar
  16. Graumann P, Marahiel MA (1996) Some like it cold: response of microorganisms to cold shock. Arch Microbiol 166:293–300. CrossRefGoogle Scholar
  17. Graumann P, Schröder K, Schmid R, Marahiel MA (1996) Cold shock stress-induced proteins in Bacillus subtilis. J Bacteriol 178:4611–4619. CrossRefGoogle Scholar
  18. Hedges AJ, Shannon R, Hobbs RP (1978) Comparison of the precision obtained in counting viable bacteria by the spiral plate maker, the droplette and the Miles and Misra Methods. J Appl Bacteriol 45:57–65. CrossRefGoogle Scholar
  19. Hellemans J, Mortier G, De Paepe A et al (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8:R19. CrossRefGoogle Scholar
  20. Hesami S, Metcalf DS, Lumsden JS, MacInnes JI (2011) Identification of cold-temperature-regulated genes in Flavobacterium psychrophilum. Appl Environ Microbiol 77:1593–1600. CrossRefGoogle Scholar
  21. Huggett J, Dheda K, Bustin S, Zumla A (2005) Real-time RT-PCR normalisation; strategies and considerations. Genes Immun 6:279–284. CrossRefGoogle Scholar
  22. Hughes KA, Cowan DA, Wilmotte A (2015) Protection of Antarctic microbial communities “out of sight, out of mind". Front Microbiol 6:151. CrossRefGoogle Scholar
  23. Jones PG, VanBogelen RA, Neidhardt FC (1987) Induction of proteins in response to low temperature in Escherichia coli. J Bacteriol 169:2092–2095. CrossRefGoogle Scholar
  24. Kozera B, Rapacz M (2013) Reference genes in real-time PCR. J Appl Genet 54:391–406. CrossRefGoogle Scholar
  25. Liu S, Meng C, Xu G et al (2018) Validation of reference genes for reverse transcription real-time quantitative PCR analysis in the deep-sea bacterium Shewanella psychrophila WP2. FEMS Microbiol Lett. Google Scholar
  26. López NI, Pettinari MJ, Stackebrandt E et al (2009) Pseudomonas extremaustralis sp. nov., a poly(3-hydroxybutyrate) producer isolated from an antarctic environment. Curr Microbiol 59:514–519. CrossRefGoogle Scholar
  27. Ma Y, Sun X, Xu X et al (2015) Investigation of reference genes in Vibrio parahaemolyticus for gene expression analysis using quantitative RT-PCR. PLoS ONE 10:e0144362. CrossRefGoogle Scholar
  28. Martínez-Rosales C, Castro-Sowinski S (2011) Antarctic bacterial isolates that produce cold-active extracellular proteases at low temperature but are active and stable at high temperature. Polar Res 30:7123. CrossRefGoogle Scholar
  29. Martínez-Rosales C, Fullana N, Musto H, Castro-Sowinski S (2012) Antarctic DNA moving forward: genomic plasticity and biotechnological potential. FEMS Microbiol Lett 331:1–9. CrossRefGoogle Scholar
  30. McMillan M, Pereg L (2014) Evaluation of reference genes for gene expression analysis using quantitative RT-PCR in Azospirillum brasilense. PLoS ONE 9:1–8. CrossRefGoogle Scholar
  31. Metcalf D, Sharif S, Weese JS (2010) Evaluation of candidate reference genes in Clostridium difficile for gene expression normalization. Anaerobe 16:439–443. CrossRefGoogle Scholar
  32. Michel V, Lehoux I, Depret G et al (1997) The cold shock response of the psychrotrophic bacterium Pseudomonas fragi involves four low-molecular-mass nucleic acid-binding proteins. J Bacteriol 179:7331–7342. CrossRefGoogle Scholar
  33. Pagliarulo V, George B, Beil SJ et al (2004) Sensitivity and reproducibility of standardized-competitive RT-PCR for transcript quantification and its comparison with real time RT-PCR. Mol Cancer 3:5. CrossRefGoogle Scholar
  34. Pessoa DDV, Vidal MS, Baldani JI, Simoes-Araujo JL (2016) Validation of reference genes for RT-qPCR analysis in Herbaspirillum seropedicae. J Microbiol Methods 127:193–196. CrossRefGoogle Scholar
  35. Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (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. CrossRefGoogle Scholar
  36. Piette F, Leprince P, Feller G (2012) Is there a cold shock response in the Antarctic psychrophile Pseudoalteromonas haloplanktis? Extremophiles 16:681–683. CrossRefGoogle Scholar
  37. Reddy GSN (2004) Psychrophilic pseudomonads from Antarctica: Pseudomonas antarctica sp. nov., Pseudomonas meridiana sp. nov. and Pseudomonas proteolytica sp. nov. Int J Syst Evol Microbiol 54:713–719. CrossRefGoogle Scholar
  38. Reiter L, Kolstø A-B, Piehler AP (2011) Reference genes for quantitative, reverse-transcription PCR in Bacillus cereus group strains throughout the bacterial life cycle. J Microbiol Methods 86:210–217. CrossRefGoogle Scholar
  39. Roberts ME, Inniss WE (1992) The synthesis of cold shock proteins and cold acclimation proteins in the psychrophilic bacterium Aquaspirillum arcticum. Curr Microbiol 25:275–278. CrossRefGoogle Scholar
  40. Rocha DJP, Santos CS, Pacheco LGC (2015) Bacterial reference genes for gene expression studies by RT-qPCR: survey and analysis. Antonie Van Leeuwenhoek 108:685–693. CrossRefGoogle Scholar
  41. Rodrigues DF, Tiedje JM (2008) Coping with our cold planet. Appl Environ Microbiol 74:1677–1686. CrossRefGoogle Scholar
  42. Russell NJ, Harrisson P, Johnston IA et al (1990) Cold adaptation of microorganisms. Philos Trans R Soc B Biol Sci 326:595–611. CrossRefGoogle Scholar
  43. Savli H (2003) Expression stability of six housekeeping genes: a proposal for resistance gene quantification studies of Pseudomonas aeruginosa by real-time quantitative RT-PCR. J Med Microbiol 52:403–408. CrossRefGoogle Scholar
  44. Smith A, Lovelace AH, Kvitko BH (2018) Validation of RT-qPCR approaches to monitor Pseudomonas syringae gene expression during infection and exposure to pattern-triggered immunity. Mol Plant-Microbe Interact 31:410–419. CrossRefGoogle Scholar
  45. Spiers AJ, Buckling A, Rainey PB (2000) The causes of Pseudomonas diversity. Microbiology 146:2345–2350. CrossRefGoogle Scholar
  46. Sumby KM, Grbin PR, Jiranek V (2012) Validation of the use of multiple internal control genes, and the application of real-time quantitative PCR, to study esterase gene expression in Oenococcus oeni. Appl Microbiol Biotechnol 96:1039–1047. CrossRefGoogle Scholar
  47. Takle GW, Toth IK, Brurberg MB (2007) Evaluation of reference genes for real-time RT-PCR expression studies in the plant pathogen Pectobacterium atrosepticum. BMC Plant Biol 7:50. CrossRefGoogle Scholar
  48. Taylor S, Wakem M, Dijkman G et al (2010) A practical approach to RT-qPCR—publishing data that conform to the MIQE guidelines. Methods 50:S1–S5. CrossRefGoogle Scholar
  49. Vandesompele J, De Preter K, Pattyn F et al (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:RESEARCH0034. CrossRefGoogle Scholar
  50. Weber MHW, Marahiel MA (2003) Bacterial cold shock responses. Sci Prog 86:9–75. CrossRefGoogle Scholar
  51. Zhang C, Xue C, Shen Y, Lu W (2015) Selection of reference genes in Saccharopolyspora spinosa for real-time PCR. Trans Tianjin Univ 21:461–467. CrossRefGoogle Scholar

Copyright information

© Springer Japan KK, part of Springer Nature 2019

Authors and Affiliations

  • César X. García-Laviña
    • 1
  • Susana Castro-Sowinski
    • 1
  • Ana Ramón
    • 1
    Email author
  1. 1.Sección Bioquímica, Departamento de Biología Celular y Molecular, Facultad de CienciasUniversidad de la RepúblicaMontevideoUruguay

Personalised recommendations