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

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 su...

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Bibliographic Details
Published in:Molecular biology (New York) 2014-11, Vol.48 (6), p.813-822
Main Authors: Sharath Chandra, G., Asokan, R., Manamohan, M., Krishna Kumar, N. K., Sita, T.
Format: Article
Language:English
Subjects:
Biochemistry
Biomedical and Life Sciences
Gene expression
Genomics. Transcriptomics
Helicoverpa armigera
Human Genetics
Insects
Life Sciences
Molecular biology
Polymerase chain reaction
Ribonucleic acid
RNA
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Summary: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 whereas, 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 .
ISSN:0026-8933
1608-3245
DOI:10.1134/S0026893314060156