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Vol 48(2014) N 6 p. 813-822; DOI 10.1134/S0026893314060156 Full Text

G. Sharath Chandra1*, R. Asokan1, M. Manamohan1, N.K. Krishna Kumar2, T. Sita3

Evaluation of reference genes for quantitative real-time PCR normalization in cotton bollworm Helicoverpa armigera

1Division of Biotechnology, Indian Institute of Horticultural Research (IIHR), Hesaraghatta Lake (PO), Bengaluru, 560 089, India
2Division of Horticulture, Krishi Anusandhan Bhawan, II, New Delhi, 110 012, India
3Department of Biotechnology, St. Martin's Engineering College, Kompally, Hyderabad, 500 030, India

*sharathgsc@gmail.com
Received - 2014-04-24; Accepted - 2014-06-02

Reverse-transcription quantitative real-time PCR (RT-qPCR), a sensitive technique is being extensively employed in quantification of gene expression. However this requires normalization with suitable reference gene (RG) which is crucial in minimizing inter sample variations. Information regarding suitable RG is scarce in general and more so in insects, including the cotton bollworm, Helicoverpa armigera, an economically important pest. In management of this pest RNA interference (RNAi) is perceived as a potential tool, which is achieved by double-stranded RNA (dsRNA) delivery. These studies demand accurate quantification of gene silencing. In this study we assessed the suitability of five RGs viz. β-actin (ACTB), 18S rRNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin (TUB) and elongation fator-1-alfa (EF1-α) for gene expression studies in dsRNA treatment and across different developmental stages of H. armigera and ranked using geNorm, NormFinder and BestKeeper software programs. Data analysis revealed that best ranked RGs were varied in dsRNA treatment and in developmental stages. Under dsRNA treatment, 18S and GAPDH were more stable hereas, TUB and GAPDH were more stable across developmental stages. We also demonstrate that inappropriate selection of RG led to erroneous estimation of the target gene, chymotrypsin expression. These results facilitate accurate quantification of gene expression in H. armigera.

Helicoverpa armigera, RNA interference, gene expression, real time PCR, reference gene, normalization



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