Plant Molecular Biology Reporter

, Volume 27, Issue 3, pp 315–320

Arbitrary Multi-gene Reference for Normalization of Real-Time PCR Gene Expression Data

  • Helen H. Tai
  • Gregory Conn
  • Charlotte Davidson
  • H. W. (Bud) Platt
Article

Abstract

Analysis of gene expression using real-time reverse transcription polymerase chain reaction (RT-PCR) requires reference genes to normalize expression values between samples. We have developed a novel reference for real-time RT-PCR using an arbitrary primer to amplify a random set of genes. The arbitrary primer amplifies over 30 genes, whose cumulative expression as measured by real-time RT-PCR closely follows that of UBQ 11, an Arabidopsis thaliana gene that is used as a reference on microarrays. The expression of arbitrary genes is also compared with potato (Solanum tuberosum spp. tubersosum) housekeeping genes and was shown to be stable during Phytophthora infestans infection.

Keywords

Arbitrary primers Gene expression Real-time PCR Reference genes 

Abbreviations

DNA

deoxyribonucleic acid

MoMLV

Moloney murine leukemia virus

RNA

ribonucleic acid

RT-PCR

reverse transcription polymerase chain reaction

DDRT-PCR

differential display reverse-transcription polymerase chain reaction

References

  1. Bower NI, Moser RJ, Hill JR, Lehnert SA (2007) Universal reference method for real-time PCR gene expression analysis of preimplantation embryos. Biotechniques 42:199–206 doi:10.2144/000112314 PubMedCrossRefGoogle Scholar
  2. Bustin SA, Benes V, Nolan T, Pfaffl MW (2005) Quantitative real-time RT-PCR—a perspective. J Mol Endocrinol 34:597–601 doi:10.1677/jme.1.01755 PubMedCrossRefGoogle Scholar
  3. Caetano-Anolles G (1993) Amplifying DNA with arbitrary oligonucleotide primers. PCR Methods Appl 3:85–94PubMedGoogle Scholar
  4. Czechowski T, Bari RP, Stitt M, Scheible WR, Udvardi MK (2004) Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root- and shoot-specific genes. Plant J 38:366–379 doi:10.1111/j.1365-313X.2004.02051.x PubMedCrossRefGoogle Scholar
  5. Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139:5–17 doi:10.1104/pp.105.063743 PubMedCrossRefGoogle Scholar
  6. Dallas PB, Gottardo NG, Firth MJ, Beesley AH, Hoffmann K, Terry PA et al (2005) Gene expression levels assessed by oligonucleotide microarray analysis and quantitative real-time RT-PCR—how well do they correlate? BMC Genomics 6:59 doi:10.1186/1471-2164-6-59 PubMedCrossRefGoogle Scholar
  7. Evers D, Schweitzer C, Nicot N, Gigliotti S, Herrera MR, Hausman JF et al (2006) Two PR-1 loci detected in the native cultivated potato Solanum phureja appear differentially expressed upon challenge by late blight. Physiol Mol Plant Pathol 67:155–163 doi:10.1016/j.pmpp.2005.12.003 CrossRefGoogle Scholar
  8. Galbraith DW, Birnbaum K (2006) Global studies of cell type-specific gene expression in plants. Annu Rev Plant Biol 57:451–475 doi:10.1146/annurev.arplant.57.032905.105302 PubMedCrossRefGoogle Scholar
  9. Gallardo K, Job C, Groot SPC, Puype M, Demol H, Vandekerckhove J et al (2001) Proteomic analysis of Arabidopsis seed germination and priming. Plant Physiol 126:835–848 doi:10.1104/pp.126.2.835 PubMedCrossRefGoogle Scholar
  10. Higuchi R, Dollinger G, Walsh PS, Griffith R (1992) Simultaneous amplification and detection of specific DNA sequences. Biotechnology (NY) 10:413–417 doi:10.1038/nbt0492-413 CrossRefGoogle Scholar
  11. Liang P, Pardee AB (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257:967–971 doi:10.1126/science.1354393 PubMedCrossRefGoogle Scholar
  12. McClelland M, Mathieu-Daude F, Welsh J (1995) RNA fingerprinting and differential display using arbitrarily primed PCR. Trends Genet 11:242–246 doi:10.1016/S0168-9525(00)89058-7 PubMedCrossRefGoogle Scholar
  13. Nakabayashi K, Okamoto M, Koshiba T, Kamiya Y, Nambara E (2005) Genome-wide profiling of stored mRNA in Arabidopsis thaliana seed germination: epigenetic and genetic regulation of transcription in seed. Plant J 41:697–709 doi:10.1111/j.1365-313X.2005.02337.x PubMedCrossRefGoogle Scholar
  14. Nicot N, Hausman JF, Hoffmann L, Evers D (2005) Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot 56:2907–2914 doi:10.1093/jxb/eri285 PubMedCrossRefGoogle Scholar
  15. Provenzano M, Mocellin S (2007) Complementary techniques: validation of gene expression data by quantitative real time PCR. Adv Exp Med Biol 593:66–73 doi:10.1007/978-0-387-39978-2_7 PubMedCrossRefGoogle Scholar
  16. Quackenbush J (2002) Microarray data normalization and transformation. Nat Genet 32(Suppl):496–501 doi:10.1038/ng1032 PubMedCrossRefGoogle Scholar
  17. Radonic A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A (2004) Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun 313:856 doi:10.1016/j.bbrc.2003.11.177 PubMedCrossRefGoogle Scholar
  18. Reid K, Olsson N, Schlosser J, Peng F, Lund S (2006) An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. BMC Plant Biol 6:27 doi:10.1186/1471-2229-6-27 PubMedCrossRefGoogle Scholar
  19. Smith RD, Brown B, Ikonomi P, Schechter AN (2003) Exogenous reference RNA for normalization of real-time quantitative PCR. Biotechniques 34:88–91PubMedGoogle Scholar
  20. Tai HH, Tai GCC, Beardmore T (2005) Dynamic histone acetylation of late embryonic genes during seed germination. Plant Mol Biol 59:909–925 doi:10.1007/s11103-005-2081-x PubMedCrossRefGoogle Scholar
  21. Tai HH, Williams M, Iyengar A, Yeates J, Beardmore T (2007) Regulation of the β-hydroxyacyl ACP dehydratase gene of Picea mariana by alternative splicing. Plant Cell Rep V26:105Google Scholar
  22. Venkatesh B, Hettwer U, Koopmann B, Karlovsky P (2005) Conversion of cDNA differential display results (DDRT-PCR) into quantitative transcription profiles. BMC Genomics 6:51 doi:10.1186/1471-2164-6-51 PubMedCrossRefGoogle Scholar
  23. Wan CY, Wilkins TA (1994) A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.). Anal Biochem 223:7–12 doi:10.1006/abio.1994.1538 PubMedCrossRefGoogle Scholar
  24. Wang Y, Barbacioru C, Hyland F, Xiao W, Hunkapiller KL, Blake J et al (2006) Large scale real-time PCR validation on gene expression measurements from two commercial long-oligonucleotide microarrays. BMC Genomics 7:59 doi:10.1186/1471-2164-7-59 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Helen H. Tai
    • 1
  • Gregory Conn
    • 1
  • Charlotte Davidson
    • 1
  • H. W. (Bud) Platt
    • 2
  1. 1.Agriculture and Agri-Food CanadaPotato Research CentreFrederictonCanada
  2. 2.Agriculture and Agri-food CanadaCrops and Livestock Research CentreCharlottetownCanada

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