Plant Molecular Biology Reporter

, Volume 22, Issue 4, pp 325–337

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

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

DOI: 10.1007/BF02772676

Cite this article as:
Iskandar, H.M., Simpson, R.S., Casu, R.E. et al. Plant Mol Biol Rep (2004) 22: 325. doi:10.1007/BF02772676

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 expressionreference genesRT-qPCRsugarcane (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

Copyright information

© International Society for Plant Molecular Biology 2004

Authors and Affiliations

  • Hayati M. Iskandar
    • 1
    • 2
    • 3
    • 4
  • 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