Identification and Validation of Reference Genes for Quantitative Real-Time Polymerase Chain Reaction in Cimex lectularius

• Published in: Journal of medical entomology 2011-07, Vol.48 (4), p.947-951
• Main Authors: Mamidala, Praveen, Rajarapu, Swapna P., Jones, Susan C., Mittapalli, Omprakash
• Format: Article
• Language: English
• Subjects: adults; Aging; Analysis; Animals; bed bug; Bed bug (Cimex lectularius); Bedbugs - genetics; Bedbugs - growth & development; BestKeeper; Biological and medical sciences; Cimex lectularius; Cuticles; Developmental stages; essential genes; Fundamental and applied biological sciences. Psychology; Gene expression; Gene Expression Profiling - standards; Genes; Genes, Insect; geNorm; instars; Malpighian tubules; Medically important nuisances and vectors, pests of stored products and materials: population survey and control; messenger RNA; Midgut; NormFinder; nymphs; Ovaries; Pesticides; Polymerase chain reaction; Polymerase Chain Reaction - standards; quantitative polymerase chain reaction; reference genes; Reference Standards; reverse transcriptase polymerase chain reaction; Ribosomal proteins; Ribosomal Proteins - analysis; Ribosomal Proteins - genetics; RNA; RNA, Messenger - genetics; SHORT COMMUNICATIONS; Tubulin; Vectors. Intermediate hosts
• ISSN: 0022-2585; 1938-2928; 0022-2585
• DOI: 10.1603/ME10262

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.