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1 July 2011 Identification and Validation of Reference Genes for Quantitative Real-Time Polymerase Chain Reaction in Cimex lectularius
Praveen Mamidala, Swapna P. Rajarapu, Susan C. Jones, Omprakash Mittapalli
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Abstract

Quantitative real-time polymerase chain reaction (qRT-PCR) has emerged as robust methodology for gene expression studies, but reference genes are crucial for accurate normalization. Commonly used reference genes are housekeeping genes that are thought to be nonregulated; however, their expression can be unstable across different experimental conditions. We report the identification and validation of suitable reference genes in the bed bug, Cimex lectularius, by using qRT-PCR. The expression stability of eight reference genes in different tissues (abdominal cuticle, midgut, Malpighian tubules, and ovary) and developmental stages (early instar nymphs, late instar nymphs, and adults) of pesticide-susceptible and pesticide-exposed C. lectularius were analyzed using geNorm, NormFinder, and BestKeeper. Overall expression analysis of the eight reference genes revealed significant variation among samples, indicating the necessity of validating suitable reference genes for accurate quantification of mRNA transcripts. Ribosomal protein (RPL18) exhibited the most stable gene expression across all the tissue and developmental-stage samples; α-tubulin revealed the least stability across all of the samples examined. Thus, we recommend RPL18 as a suitable reference gene for normalization in gene expression studies of C. lectularius.

© 2011 Entomological Society of America
Praveen Mamidala, Swapna P. Rajarapu, Susan C. Jones, and Omprakash Mittapalli "Identification and Validation of Reference Genes for Quantitative Real-Time Polymerase Chain Reaction in Cimex lectularius," Journal of Medical Entomology 48(4), 947-951, (1 July 2011). https://doi.org/10.1603/ME10262
Received: 3 December 2010; Accepted: 1 May 2011; Published: 1 July 2011
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KEYWORDS
bed bug
BestKeeper
geNorm
NormFinder
reference genes
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