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Background   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.    Results  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.    Conclusions  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.

bmc research notes

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