Aparajita Choudhury1, Shubham Verma2, Mehanathan Muthamilarasan2, Manchikatla Venkat Rajam3. 1. Department of Genetics, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India. 2. Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Telangana, 500046, India. 3. Department of Genetics, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India. rajam.mv@gmail.com.
Abstract
BACKGROUND: Maruca vitrata is one of the potential insect pests that cause devastating losses to legume cultivation worldwide. Gene functional studies facilitate dissecting the molecular mechanisms underlying the infection process and enable devising appropriate molecular strategies to control this insect pest. Expression profiling using quantitative real-time PCR (qRT-PCR) provides insights into the functional characterization of target genes; however, ideal reference genes should be deployed in such studies to nullify the background variation and improve the accuracy of target gene expression. An ideal reference gene should have a stable expression across developmental stages, biological conditions, tissues, or experimental conditions. METHODS AND RESULTS: Given this, the stability of eight candidate reference genes was evaluated in M. vitrata at different developmental stages, diets, and sexes by qRT-PCR method, and the data was analyzed using four independent algorithms, namely GeNorm, NormFinder, BestKeeper, and ΔCt, and one comprehensive algorithm, RefFinder. CONCLUSION: The analysis showed that RP49 and RPL13 were the best suitable reference genes for studying target gene expression at different developmental stages. Further, the study identified RP49 and RPL24, and GAPDH and RPL24 as the ideal reference genes in M. vitrata fed with different diets and sexes, respectively. The reference genes reported in the present study will ensure the accuracy of target gene expression, and thus, will serve as an important resource for gene functional studies in M. vitrata.
BACKGROUND: Maruca vitrata is one of the potential insect pests that cause devastating losses to legume cultivation worldwide. Gene functional studies facilitate dissecting the molecular mechanisms underlying the infection process and enable devising appropriate molecular strategies to control this insect pest. Expression profiling using quantitative real-time PCR (qRT-PCR) provides insights into the functional characterization of target genes; however, ideal reference genes should be deployed in such studies to nullify the background variation and improve the accuracy of target gene expression. An ideal reference gene should have a stable expression across developmental stages, biological conditions, tissues, or experimental conditions. METHODS AND RESULTS: Given this, the stability of eight candidate reference genes was evaluated in M. vitrata at different developmental stages, diets, and sexes by qRT-PCR method, and the data was analyzed using four independent algorithms, namely GeNorm, NormFinder, BestKeeper, and ΔCt, and one comprehensive algorithm, RefFinder. CONCLUSION: The analysis showed that RP49 and RPL13 were the best suitable reference genes for studying target gene expression at different developmental stages. Further, the study identified RP49 and RPL24, and GAPDH and RPL24 as the ideal reference genes in M. vitrata fed with different diets and sexes, respectively. The reference genes reported in the present study will ensure the accuracy of target gene expression, and thus, will serve as an important resource for gene functional studies in M. vitrata.
Authors: Stephen A Bustin; Vladimir Benes; Jeremy A Garson; Jan Hellemans; Jim Huggett; Mikael Kubista; Reinhold Mueller; Tania Nolan; Michael W Pfaffl; Gregory L Shipley; Jo Vandesompele; Carl T Wittwer Journal: Clin Chem Date: 2009-02-26 Impact factor: 8.327