Assessing reference genes for accurate transcript normalization using quantitative real-time PCR in pearl millet [Pennisetum glaucum (L.) R. Br]

PLoS One. 2014 Aug 29;9(8):e106308. doi: 10.1371/journal.pone.0106308. eCollection 2014.


Pearl millet [Pennisetum glaucum (L.) R.Br.], a close relative of Panicoideae food crops and bioenergy grasses, offers an ideal system to perform functional genomics studies related to C4 photosynthesis and abiotic stress tolerance. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) provides a sensitive platform to conduct such gene expression analyses. However, the lack of suitable internal control reference genes for accurate transcript normalization during qRT-PCR analysis in pearl millet is the major limitation. Here, we conducted a comprehensive assessment of 18 reference genes on 234 samples which included an array of different developmental tissues, hormone treatments and abiotic stress conditions from three genotypes to determine appropriate reference genes for accurate normalization of qRT-PCR data. Analyses of Ct values using Stability Index, BestKeeper, ΔCt, Normfinder, geNorm and RefFinder programs ranked PP2A, TIP41, UBC2, UBQ5 and ACT as the most reliable reference genes for accurate transcript normalization under different experimental conditions. Furthermore, we validated the specificity of these genes for precise quantification of relative gene expression and provided evidence that a combination of the best reference genes are required to obtain optimal expression patterns for both endogeneous genes as well as transgenes in pearl millet.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Gene Expression Regulation, Plant / physiology*
  • Genes, Plant / physiology*
  • Pennisetum / metabolism*
  • Plant Proteins / biosynthesis*
  • Plant Proteins / genetics
  • Real-Time Polymerase Chain Reaction / methods*
  • Real-Time Polymerase Chain Reaction / standards
  • Reference Standards
  • Software*


  • Plant Proteins

Grants and funding

This work is funded by The United States Agency for International Development (USAID) under the Grant No. APS M/OAA/GRO/EGAS-11-002011. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.