Quantitative real-time PCR based evaluation and validation of reference genes in Gossypium arboreum
P, RAGHAVENDRA K | KUMAR, RAKESH | DAS, JOY | B, SANTOSH H | MORE, SACHIN A | N, RAMAKRISHNA | CHAWLA, SHILPA G | KRANTHI, SANDHYA | KRANTHI, KESHAV RAJ
Estimation of gene expression levels plays a crucial role in understanding the function of the target gene(s). Intersamplevariance in gene expression can be more precisely measured if transcripts levels are accurately normalized.Normalization is pre-requisite step prior to the determination of candidate gene expression by qPCR. In this studyconducted at ICAR-Central Institute for Cotton Research, Nagpur during 2015–16, six candidate reference genes,viz. actin4 (ACT4), actin7(ACT7), RNA Helicase (RNAH), Serine/threonine-protein phosphatase PP2A-1(PP2A1),ubiquitin7 (UBQ7) and α tubulin (αTUB) were systematically analysed for their expression patterns in differenttissues pertaining to three development stages of cotton namely seedling, early reproductive and fiber development.The study has identified actin-4/actin-7/ubiquitin-7 as the most ideal reference genes for fiber development stageswhereas actin-4/ ubiquitin-7 and actin-7/RNA helicases for seedling and early reproductive development stages,respectively. Validation of identified reference genes for relative expression analysis of Gacobl9, a COBRA-likeprotein, demonstrated their usefulness in qPCR analysis in Gossypium arboreum.
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