Physiology and Molecular Biology of Plants

, Volume 25, Issue 1, pp 123–134 | Cite as

Validation of reference genes for qRT-PCR data normalisation in lentil (Lens culinaris) under leaf developmental stages and abiotic stresses

  • Ragini Sinha
  • T. R. Sharma
  • Anil Kumar SinghEmail author
Research Article


Lentil (Lens culinaris) is one of the most important staple food crops of developing countries. Transcriptome based global gene expression profiling followed by validation of expression of important genes through quantitative real time-PCR (qRT-PCR) has achieved significance in recent years. However, there is a severe scarcity of information regarding stable reference genes in lentil, which is mandatory for qRT-PCR data normalisation. Hence, the present study was under-taken to identify the most stable reference gene(s) in lentil. Expression stability of eight candidate genes viz. ribulose 1,5-bisphosphate carboxylase large subunit (Rbcl), ribosomal protein L2 (RPL2), 18S rRNA, tubulin (Tub), elongation factor 1α (EF1α), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), heat shock protein (HSP70), and Maturase (mat K) was evaluated in five varieties of lentil at three different stages of leaf development and abiotic stress conditions using qRT-PCR. The results were analysed using four types of statistical software viz., geNorm, BestKeeper, NormFinder and RefFinder; all softwares identified RPL2 as most stable under abiotic stress conditions and developmental stages followed by Tub and Rbcl; while, HSP70 was identified as least stable. Relative expression of the target genes, defensin and PR4, was evaluated under abiotic stress conditions and data normalisation was done using two stable reference genes, RPL2 and Tub, either alone or in combination and with two least stable genes, HSP70 and 18S. The present work provides a list of potential reference genes in lentil, which will help in selection of appropriate reference gene for qRT-PCR data normalization depending upon the experiment.


Abiotic stress Gene expression Growth stages Lentil (Lens culinarisReference gene qRT-PCR 



RS acknowledges Science and Engineering Research Board, Department of Science and Technology, Government of India for the National-Postdoctoral Fellowship (PDF/2016/000924). AKS acknowledges Institute projects IXX12585 and IXX12644 funded by ICAR-Indian Institute of Agricultural Biotechnology, Ranchi. We thank Dr. Madhuparna Banerjee, Associate Professor, College of Biotechnology, Birsa Agricultural University, Ranchi for granting access to her lab facilities during the course of this study.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

12298_2018_609_MOESM1_ESM.pdf (753 kb)
Supplementary material 1 (PDF 753 kb)


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Copyright information

© Prof. H.S. Srivastava Foundation for Science and Society 2018

Authors and Affiliations

  1. 1.ICAR-Indian Institute of Agricultural BiotechnologyGarhkhatanga, RanchiIndia

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