title: 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.
abstract: 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.
authors.affiliation: Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627, Pampulha, Belo Horizonte, MG, Brazil.
authors.forename: Rafaela M
authors.initials: RM
authors.lastname: Paim
authors.forename: Marcos H
authors.initials: MH
authors.lastname: Pereira
authors.forename: Raffaello
authors.initials: R
authors.lastname: Di Ponzio
authors.forename: Juliana O
authors.initials: JO
authors.lastname: Rodrigues
authors.forename: Alessandra A
authors.initials: AA
authors.lastname: Guarneri
authors.forename: Nelder F
authors.initials: NF
authors.lastname: Gontijo
authors.forename: Ricardo N
authors.initials: RN
authors.lastname: Araújo
mesh_terms: D000818:Animals; D015870:Gene Expression; D020869:Gene Expression Profiling; D020043:Genes, Essential; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000166:cytology; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards; D012015:Reference Standards; D012239:Rhodnius / Q000235:genetics* / Q000469:parasitology; D012469:Salivary Glands / Q000166:cytology / Q000378:metabolism*; D012984:Software; D014349:Trypanosoma cruzi / Q000502:physiology; D057509:Trypanosoma rangeli / Q000502:physiology; D014404:Tubulin / Q000235:genetics
chemical_list: D012337:RNA, Ribosomal, 18S; D014404:Tubulin; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)