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Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 115, Issue 1, pp 13–22 | Cite as

Reference genes for quantitative real-time PCR analysis in the model plant foxtail millet (Setaria italica L.) subjected to abiotic stress conditions

  • Karunesh Kumar
  • Mehanathan Muthamilarasan
  • Manoj PrasadEmail author
Original Paper

Abstract

Reference genes are standards for quantifying gene expression through quantitative real-time PCR (qRT-PCR); however, the variation observed in their expression levels is the major hindrance towards realising their effective use. Hence, a systematic validation of reference genes is required to ensure proper normalization. However, no such study has been conducted in foxtail millet [Setaria italica (L.)], which has recently emerged as a model crop for genetic and genomic studies. In the present study, 8 commonly used reference genes were evaluated, including 18S ribosomal RNA, elongation factor-1α, Actin2, alpha tubulin, beta tubulin, translation factor, RNA polymerase II and adenine phosphoribosyl transferase. Expression stability of candidate internal control genes was investigated under salinity and dehydration treatments. The results obtained suggested a wide range of Ct values and variable expression of all reference genes. geNorm and NormFinder analysis had revealed that Act2 and RNA POL II are suitable reference genes for salinity stress-related studies and EF- and RNA POL II are appropriate internal controls for dehydration stress-related expression analyses. These qualified reference genes has also been validated for relative quantification of 14-3-3 expression analysis which demonstrated their applicability. Thus, this is the first report on selection and validation of superior reference genes for qRT-PCR in foxtail millet under different abiotic stress conditions.

Keywords

Abiotic stress Foxtail millet Reference genes Quantitative real time PCR (qRT-PCR) GeNorm NormFinder Setaria italica 

Notes

Acknowledgments

Grateful thanks are due to the Director, National Institute of Plant Genome Research (NIPGR), New Delhi, India for providing facilities. The authors work in this area was supported by the core Grant of NIPGR. Mr. Karunesh Kumar and Mr. Mehanathan Muthamilarasan acknowledge the award of Senior Research Fellowship and Junior Research Fellowship from Council of Scientific and Industrial Research and University Grants Commission, New Delhi, India, respectively.

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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Karunesh Kumar
    • 1
  • Mehanathan Muthamilarasan
    • 1
  • Manoj Prasad
    • 1
    Email author
  1. 1.National Institute of Plant Genome Research (NIPGR)New DelhiIndia

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