Validation of reference genes for expression analysis in the salivary gland and the intestine of Rhodnius prolixus (Hemiptera, Reduviidae) under different experimental conditions by quantitative real-time PCR.
Validation of reference genes for expression analysis in the salivary gland and the intestine of Rhodnius prolixus (Hemiptera, Reduviidae) under different experimental conditions by quantitative real-time PCR.
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DOI：
10.1186/1756-0500-5-128
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发表时间：
2012-03-06
影响因子：
1.8
通讯作者：
Araújo RN
中科院分区：
文献类型：
--
作者：
Paim RM;Pereira MH;Di Ponzio R;Rodrigues JO;Guarneri AA;Gontijo NF;Araújo RN
长角锥虫是一种能将克氏锥虫和兰氏锥虫传播给脊椎动物宿主的吸血昆虫。近年来，人类病原体的无脊椎媒介的基因组资源显著增加，长尾霉是三足动物中研究的主要物种之一。然而，关于这个物种的许多基本分子方面的信息的缺乏限制了可用的基因组信息的使用。本研究旨在通过确定最合适的内参基因来规范qPCR的mRNA表达数据，从而促进基因表达研究。采用qPCR方法评价5个候选内参基因（18S rRNA、GAPDH、β-肌动蛋白、α-微管蛋白和核糖体蛋白L26）在两种组织（唾腺和肠道）和不同生理条件下（血喂养前后和感染克氏T.或朗格里T.后）的表达稳定性。使用geNorm、NormFinder和BestKeeper三种软件程序对结果进行分析。所有被评估的候选基因都被证明可以作为参考基因，但根据实验条件，有些基因被认为更合适。18S、GAPDH和α-微管蛋白在所有组织和实验条件下均表现出可接受的稳定性。β-肌动蛋白（β-actin）是应用最广泛的内参基因之一，在唾液腺的研究中被证实是最合适的内参基因之一，但在昆虫吸血后的肠道中表达稳定性最低。在研究中，L26被确定为最差的内参基因。在不同的组织样品和不同的实验条件下，基因的表达稳定性是不同的。三个统计软件包提供的结果强调了所有五个测试的参考基因在作物和唾液腺中的适用性，只有少数例外。这一结果强调了验证内参基因对长尾草qRT-PCR分析的重要性。
Rhodnius prolixus is a blood-feeding insect that can transmit Trypanosoma cruzi and Trypanosoma rangeli to vertebrate hosts. Recently, genomic resources for invertebrate vectors of human pathogens have increased significantly, and R. prolixus has been one of the main species studied among the triatomines. However, the paucity of information on many of the fundamental molecular aspects of this species limits the use of the available genomic information. The present study aimed to facilitate gene expression studies by identifying the most suitable reference genes for the normalization of mRNA expression data from qPCR. The expression stability of five candidate reference genes (18S rRNA, GAPDH, β-actin, α-tubulin and ribosomal protein L26) was evaluated by qPCR in two tissues (salivary gland and intestine) and under different physiological conditions: before and after blood feeding and after infection with T. cruzi or T. rangeli. The results were analyzed with three software programs: geNorm, NormFinder and BestKeeper. All of the evaluated candidate genes proved to be acceptable as reference genes, but some were found to be more appropriate depending on the experimental conditions. 18S, GAPDH and α-tubulin showed acceptable stability for studies in all of the tissues and experimental conditions evaluated. β-actin, one of the most widely used reference genes, was confirmed to be one of the most suitable reference genes in studies with salivary glands, but it had the lowest expression stability in the intestine after insect blood feeding. L26 was identified as the poorest reference gene in the studies performed. The expression stability of the genes varies in different tissue samples and under different experimental conditions. The results provided by three statistical packages emphasize the suitability of all five of the tested reference genes in both the crop and the salivary glands with a few exceptions. The results emphasise the importance of validating reference genes for qRT-PCR analysis in R. prolixus studies.