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

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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.

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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

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Authors and Affiliations

  1. School of Molecular and Microbial Sciences, University of Queensland, 4067, St Lucia, Queensland, Australia

    Hayati M. Iskandar, Robert S. Simpson & Donald J. Maclean

  2. CSIRO, Plant Industry, Queensland Bioscience Precinct, 306 Carmody Road, 4067, St Lucia, Queensland, Australia

    Hayati M. Iskandar, Rosanne E. Casu, Graham D. Bonnett & John M. Manners

  3. Cooperative Research Centre for Sugar Industry Innovation through Biotechnology, University of Queensland, 4072, St Lucia, Queensland, Australia

    Hayati M. Iskandar, Rosanne E. Casu, Graham D. Bonnett & John M. Manners

  4. Indonesian Biotechnology Research Institute for Estate Crops, J1. Taman Kencana, No. 1, 16151, Bogor, Indonesia

    Hayati M. Iskandar

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  1. Hayati M. Iskandar
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  2. Robert S. Simpson
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  3. Rosanne E. Casu
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  4. Graham D. Bonnett
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  5. Donald J. Maclean
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  6. John M. Manners
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Correspondence to Hayati M. Iskandar.

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Iskandar, H.M., Simpson, R.S., Casu, R.E. et al. Comparison of reference genes for quantitative real-time polymerase chain reaction analysis of gene expression in sugarcane. Plant Mol Biol Rep 22, 325–337 (2004). https://doi.org/10.1007/BF02772676

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  • DOI: https://doi.org/10.1007/BF02772676

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