Advertisement

Plant Molecular Biology Reporter

, Volume 22, Issue 4, pp 325–337 | Cite as

Comparison of reference genes for quantitative real-time polymerase chain reaction analysis of gene expression in sugarcane

  • Hayati M. IskandarEmail author
  • Robert S. Simpson
  • Rosanne E. Casu
  • Graham D. Bonnett
  • Donald J. Maclean
  • John M. Manners
Commentary

Abstract

A protocol for reverse transcription followed by real-time quantitative PCR (RT-qPCR) analysis of tissue-specific and genotype-variable gene expression in sugarcane (Saccharum sp.) was developed. A key requirement for this analysis was the identification of a housekeeping gene with transcript levels that were relatively stable across tissues and genotypes, suitable for use as a reference. Primers for β-actin, β-tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes and 25S ribosomal RNA were designed and tested by RT-qPCR, and formation of product in the reactions was measured with the SYBR green I dye system. Ribosomal RNA was the most sensitive and consistent as a reference gene. Determination of the expression levels of β-actin, β-tubulin, and GAPDH transcripts relative to that of 25S rRNA showed that GAPDH had the most consistent mRNA expression of protein-coding genes across different tissues. GAPDH also showed low variation in expression in maturing stem internodes when compared across 2 cultivars and 3 otherSaccharum species. GAPDH therefore appears to be a suitable “housekeeping gene” in addition to 25S rRNA as a reference for measuring the relative expression of other genes in sugarcane. With use of GAPDH as a reference, the relative expression of the sugarcane sugar transporter genePst2a was assessed in a range of tissues. The result obtained was similar to our previously published Northern blot analysis. The protocol described here, using GAPDH as a reference gene, is recommended for studying the expression of other genes of interest in diverse tissues and genotypes of sugarcane.

Key words

gene expression reference genes RT-qPCR sugarcane (Saccharum

Abbreviations

DEPC

diethyl pyrocarbonate

EST

expressed sequence tag

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

rRNA

ribosomal RNA

RT-qPCR

reverse transcription followed by quantitative real-time PCR

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Aitken KS, Jackson PA, and McIntyre CL (in press) A combination of AFLP and SSR markers provides extensive map coverage and identification of homo(eo)logous linkage groups in a sugarcane cultivar. Theor Appl Genet.Google Scholar
  2. Altschul SF, Gish W, Miller W, Meyers EW, and Lipman DJ (1990) Basic local alignmen search tool. J Mol Biol 215: 403–410.PubMedGoogle Scholar
  3. Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25: 169–193PubMedCrossRefGoogle Scholar
  4. Carson DL and Botha FC (2000) Preliminary analysis of expressed sequence tags for sugarcane. Crop Sci 40: 1769–1779.CrossRefGoogle Scholar
  5. Casu RE, Grof CPL, Rae AL, McIntyre CL, Dimmock CM, and Manners JM (2003) Identification of a novel sugar transporter homologue strongly expressed in maturing stem vascular tissues of sugarcane by expressed sequence tag and microarray analysis. Plant Mol Biol 52: 371–386.PubMedCrossRefGoogle Scholar
  6. Casu RE, Dimmock CM, Chapman SC, Grof CPL, McIntyre CL, Bonnett GD, and Manners JM (2004) Identification of differentially expressed transcripts from maturing stem of sugarcane byin silico analysis of stem expressed sequence tags and gene expression profiling. Plant Mol Biol 54: 503–517.PubMedCrossRefGoogle Scholar
  7. Czechowski T, Bari RP, Stitt M, Scheible W, and Udvardi MK (2004) Real-time RT-PCR profiling of over 1400Arabidopsis transcription factors: unprecedented sensitivity reveals novel root- and shoot-specific genes. Plant J 38: 366–379.PubMedCrossRefGoogle Scholar
  8. Edwards KJ (2004) Performing real-time PCR. In: Edwards K, Logan J, and Saunders N (eds), Real-Time PCR: An Essential Guide, Horizon Bioscience, Hethersett, Norwich, UK, pp 71–83.Google Scholar
  9. Gachon C, Mingam A, and Charrier B (2004) Real-time PCR: what relevance to plant studies? J Exp Bot 55(402): 1445–1454.PubMedCrossRefGoogle Scholar
  10. Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, and Mathieu C (2001) An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods 25: 386–401.PubMedCrossRefGoogle Scholar
  11. Grivet L and Arruda P (2001) Sugarcane genomics: depicting the complex genome of an important tropical crop. Curr Opin Plant Biol 5: 122–127.CrossRefGoogle Scholar
  12. Kim B-R, Nam H-Y, Kim S-U, Kim S-I, and Chang Y-J (2003) Normalization of reverse transcription quantitative-PCR with housekeeping genes in rice. Biotechnol Lett 25: 1869–1872.PubMedCrossRefGoogle Scholar
  13. Klok EJ, Wilson IW, Wilson D, Chapman SC, Ewing RM, Somerville SC, Peacock WJ, Dolferus R, and Dennis ES (2002) Expression profile analyses of the low-oxygen response inArabidopsis root cultures. Plant Cell 14: 2481–2494.PubMedCrossRefGoogle Scholar
  14. Nogueira FTS, De Rosa VE, Menossi M, Ulian EC, and Arruda P (2003) RNA expression profiles and data mining of sugarcane response to cold temperature. Plant Physiol 132: 1811–1824.PubMedCrossRefGoogle Scholar
  15. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29: 2002–2007.CrossRefGoogle Scholar
  16. Ramakers C, Ruijter JM, Deprez RHL, and Moorman AFM (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339: 62–66.PubMedCrossRefGoogle Scholar
  17. Saunders NA (2004) Quantitative real time PCR. In: Edwards K, Logan J, and Saunders N (eds), Real-Time PCR: An Essential Guide, Horizon Bioscience, Hethersett, Norwich, UK, pp 103–123.Google Scholar
  18. Sambrook J, Fritsch EF, and Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  19. Schenk PM, Kazan K, Manners JM, Anderson JP, Simpson RS, Wilson IW, Somerville SC, and Maclean DJ (2003) Systemic gene expression inArabidopsis during an incompatible interaction withAlternaria brasicicola. Plant Physiol 132: 999–1010.PubMedCrossRefGoogle Scholar
  20. Vettore AL, da Silva FR, Kemper EL, and Arruda P (2001) The libraries that made SUCEST. Genet Mol Biol 24: 1–7.CrossRefGoogle Scholar
  21. Zou J-W, Sun M-X, and Yang H-Y (2002) Single-embryo RT-PCR assay to study gene expression dynamics during embryogenesis inArabidopsis thaliana. Plant Mol Biol Rep 20: 19–26.CrossRefGoogle Scholar

Copyright information

© International Society for Plant Molecular Biology 2004

Authors and Affiliations

  • Hayati M. Iskandar
    • 1
    • 2
    • 3
    • 4
    Email author
  • Robert S. Simpson
    • 1
  • Rosanne E. Casu
    • 2
    • 3
  • Graham D. Bonnett
    • 2
    • 3
  • Donald J. Maclean
    • 1
  • John M. Manners
    • 2
    • 3
  1. 1.School of Molecular and Microbial SciencesUniversity of QueenslandSt LuciaAustralia
  2. 2.CSIRO, Plant IndustryQueensland Bioscience PrecinctSt LuciaAustralia
  3. 3.Cooperative Research Centre for Sugar Industry Innovation through BiotechnologyUniversity of QueenslandSt LuciaAustralia
  4. 4.Indonesian Biotechnology Research Institute for Estate CropsBogorIndonesia

Personalised recommendations