Gene expression analysis using quantitative real-time PCR (qRTPCR) is a very sensitive technique which completely depends on stable performance of reference genes used in the study. 3 July 2014

1. Inclusive Market-Oriented Development (IMOD) –
our approach to bringing prosperity in the drylands.
ICRISAT is a member of the CGIAR Consortium.
Identification of perfect housekeeping genes
for gene expression studies in pigeonpea by
quantitative real-time PCR
Pallavi Sinha, Rachit K. Saxena, Vikas K. Singh, V. Suryanarayana, L. Krishnamurthy,
Rajeev K. Varshney*
International Crop Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, India
*Address of corresponding author: r.k.varshney@cgiar.org
Gene expression analysis using quantitative real-time PCR (qRT-PCR) is a very sensitive technique which completely
depends on stable performance of reference genes used in the study. A number of housekeeping genes have been used as
reference genes in various expression studies however, they found to be varying under different stress conditions. To identify
the suitable housekeeping genes in pigeonpea for expression analysis under different abiotic stress conditions (drought, heat
and salinity) we have compared relative expression variations of 10 commonly used housekeeping genes (EF1α, UBQ10,
GAPDH, 18srRNA, 25srRNA, TUB6, ACT1, IF4α, UBC and HSP90) on various tissues (root, stem and leaves) of Asha (ICPL
87119). Based on analysis of drought stress conditions IF4α followed by TUB6 and HSP90 were identified as most stable
reference genes. For heat stress conditions EF1α, UBC and HSP90 were found most stable reference genes. Similarly, for
salinity stress conditions, GAPDH followed by UBC and ACT1 were identified as the most stable reference genes. Stable and
most unstable genes identified in the study were validated to estimate the expression level of candidate gene in pigeonpea
(Cc_cds_33874) for drought tolerance and results obtained confirm the ranking of the reference genes identified in the study.
Abstract
Gene
name
Gene description
EF1α Elongation factor Tu GTP binding
domain
UBQ10 Ubiquitin family
GAPDH Glyceraldehyde 3-phosphate
dehydrogenase
18SrRNA 18S ribosomal RNA
25SrRNA 25S ribosomal RNA
TUB6 Tubulin/FtsZ family, GTPase domain
ACT1 Actin 1
IF4α Initiation factor 4a
UBC Ubiquitin-conjugating enzyme
HSP90 Heat shock protein 90
Approach Gene expression stability graph
Financial support from USAID is gratefully acknowledged. This
work has been undertaken as part of the CGIAR Research
Program on Grain Legumes.
Summary
The present study revealed that for drought stress condition IF4α
followed by TUB6 and HSP90, for heat stress condition EF1α and
HSP90 and for salinity stress GAPDH followed by UBC and ACT1
were the most stable reference genes. Therefore, our study will
facilitate selection of the suitable housekeeping genes for
transcript profiling using qRT-PCR for specific abiotic stress
condition for functional genomics studies in pigeonpea .
Drought
Vegetative /
reproductive
Heat
Vegetative /
reproductive
Salinity
Vegetative /
reproductive
cDNA synthesis
qRT-PCR
Gene stability Analysis
RNA isolation
Root, shoot and leaves were collected
Stresses
/Stages
Asha
Gene Validation
Stresses
/Stages
Acknowledgement
Validation of identified genes
Trait Drought Heat Salinity
Genes/ Tools geNorm NormFinder geNorm NormFinder geNorm NormFinder
M-
value
Rank S-
value
Rank M-
value
Rank S- value Rank M-
value
Rank S- value Rank
EF1α 0.924 7 0.043 6 0.000 1 0.015 1 0.555 5 0.723 7
UBQ10 0.842 6 0.055 8 1.486 9 0.142 8 0.650 6 0.643 6
GAPDH 0.757 4 0.034 5 0.575 3 0.028 4 0.384 1 0.192 1
18srRNA 1.193 9 0.127 10 1.090 7 0.151 9 0.943 8 1.345 9
25srRNA 1.090 8 0.122 9 1.196 8 0.153 10 0.819 7 1.179 8
TUB6 0.504 1 0.032 4 0.934 6 0.061 7 1.046 9 1.353 10
ACT1 0.682 3 0.029 3 0.702 4 0.029 5 0.505 4 0.491 3
IF4α 0.504 1 0.027 1 0.840 5 0.048 6 0.474 3 0.613 5
UBC 0.566 2 0.049 7 0.419 2 0.015 3 0.384 1 0.210 2
HSP90 0.786 5 0.028 2 0.000 1 0.015 2 0.422 2 0.544 4
Results: Ranking of genes based on stability
Plant tissues
Foldexpressionlevel