Identification and Validation of Reference Genes for Quantitative Real-Time Polymerase Chain Reaction in Cimex lectularius
Identification and Validation of Reference Genes for Quantitative Real-Time Polymerase Chain Reaction in Cimex lectularius
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DOI：
10.1603/me10262
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发表时间：
2011-07-01
影响因子：
2.1
通讯作者：
Mittapalli, Omprakash
中科院分区：
文献类型：
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作者：
Mamidala, Praveen;Rajarapu, Swapna P.;Mittapalli, Omprakash
定量实时聚合酶链式反应(qRT-PCR)已成为基因表达研究的可靠方法，但参考基因对于准确的标准化至关重要。常用的参考基因是被认为不受调控的管家基因；然而，它们的表达在不同的实验条件下可能是不稳定的。我们报道了利用qRT-PCR技术在臭虫Cimex lectularius中鉴定和验证合适的参考基因。利用geNorm、NormFinder和BestKeeper分析了8个参考基因在不同发育阶段(早龄若虫、晚龄若虫和成虫)的不同组织(腹部角质层、中肠、马氏管和卵巢)中的表达稳定性。对八个参考基因的总体表达分析显示，样本之间存在显著差异，这表明有必要验证合适的参考基因，以准确量化mRNA转录本。核糖体蛋白(RPL18)在所有组织和发育阶段的样本中显示出最稳定的基因表达；α-微管蛋白在所有被检查的样本中显示出最不稳定的基因表达。因此，我们推荐RPL18作为研究华支睾吸虫基因表达的一个合适的参考基因。
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; alpha-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.