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Comprehensive evaluation of candidate reference genes for real-time quantitative PCR (RT-qPCR) data normalization in nutri-cereal finger millet [Eleusine Coracana (L.)]

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Relation http://oar.icrisat.org/10898/
http://dx.doi.org/10.1371/journal.pone.0205668
10.1371/journal.pone.0205668
 
Title Comprehensive evaluation of candidate reference genes for real-time quantitative PCR (RT-qPCR) data normalization in nutri-cereal finger millet [Eleusine Coracana (L.)]
 
Creator Reddy, P S
Dhaware, M G
Reddy, D S
Reddy, B P
Divya, K
Sharma, K K
Bhatnagar-Mathur, P
 
Subject Millets
Finger Millet
Genetics and Genomics
Food and Nutrition
 
Description Finger millet (Eleusine coracana L.) is an annual herbaceous self-pollinating C4 cereal crop of the arid and semi-arid regions of the world. Finger millet is a food security crop proven to have resilience to changing climate and scores very high in nutrition. In the current study, we have assessed sixteen candidate reference genes for their appropriateness for the normalization studies in finger millet subjected to experimental regimes and treatments. Ten candidate reference genes (GAPDH, β-TUB, CYP, EIF4α, TIP41, UBC, G6PD, S24, MACP and MDH) were cloned and six (ACT, ELF1α, PP2A, PT, S21 and TFIID) were mined from the NCBI database as well as from the literature. Expression stability ranking of the finger millet reference genes was validated using four different statistical tools i.e., geNorm, NormFinder, BestKeeper, ΔCt and RefFinder. From the study, we endorse MACP, CYP, EIF4α to be most stable candidate reference genes in all ‘tissues’, whereas PT, TFIID, MACP ranked high across genotypes, β-TUB, CYP, ELF1α were found to be best under abiotic stresses and ‘all samples set’. The study recommends using minimum of two reference genes for RT-qPCR data normalizations in finger millet. All in all, CYP, β-TUB, and EF1α, in combination were found to be best for robust normalizations under most experimental conditions. The best and the least stable genes were validated for confirmation by assessing their appropriateness for normalization studies using EcNAC1 gene. The report provides the first comprehensive list of suitable stable candidate reference genes for nutritional rich cereal finger millet that will be advantageous to gene expression studies in this crop.
 
Publisher Public Library of Science
 
Date 2018-10-15
 
Type Article
PeerReviewed
 
Format application/pdf
 
Language en
 
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Identifier http://oar.icrisat.org/10898/1/Comprehensive%20evaluation%20of%20candidate.pdf
Reddy, P S and Dhaware, M G and Reddy, D S and Reddy, B P and Divya, K and Sharma, K K and Bhatnagar-Mathur, P (2018) Comprehensive evaluation of candidate reference genes for real-time quantitative PCR (RT-qPCR) data normalization in nutri-cereal finger millet [Eleusine Coracana (L.)]. PLoS One (TSI), 13 (10). pp. 1-17. ISSN 1932-6203