Russian Journal of Plant Physiology

, Volume 65, Issue 6, pp 890–897 | Cite as

Validation of Appropriate Reference Genes for Real-Time Quantitative PCR Gene Expression Analysis in Rice Plants Exposed to Metal Stresses

  • D. Ebadi AlmasEmail author
  • A. Rahmani Kamrodi
Research Papers


Environmental pollution by toxic heavy metals may lead to the possible contamination of the rice plant (Oryza sativa L.). Although gene expression analysis through real-time quantitative PCR (RT-qPCR) has increased our knowledge about biological responses to heavy metals, gene network that mediates rice plant responses to heavy metal stress remains elusive. In such scenario, validation of reference gene is a major requirement for successful analyzes involving RT-qPCR. In this study, we analyzed the expression stability of eight commonly used housekeeping genes (GAPDH, Actin, eIF-4α, UBQ 5, UBQ 10, UBC, EF-1α and β-TUB) in rice leaves exposed to four kinds of heavy metals (Zn, Cu, Cd and Pb). The expression stability of these genes was determined using geNorm, NormFinder, BestKeeper and RefFinder algorithms. The results showed that UBQ 10 and UBC were the most stable reference genes across all the tested samples. We measured the expression profiles of the heavy metal-inducible gene O. sativa METALLOTHIONEIN2b (OsMT2b) using the two most stable and one least stable reference genes in all samples. The relative expression of OsMT2b varied greatly according to the different reference genes. Our results may be beneficial for future studies involving the quantification of relative gene expression levels in rice plants.


Oryza sativa reference gene RT-qPCR metal stress 





cycle threshold


eukaryotic elongation factor 1-alpha


eukaryotic-initiation factor 4α


glyceraldehyde-3-phosphate dehydrogenase


stability value




standard deviation


ubiquitin conjugating enzyme E2


ubiquitin 5

UBQ 10

ubiquitin 10

Vn/Vn + 1

pairwise variation




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Copyright information

© Pleiades Publishing, Ltd. 2018

Authors and Affiliations

  1. 1.Plant Breeding and Biotechnology Department, Faculty of Plant ProductionAgricultural Sciences and Natural Resources UniversityGorganIran

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