BackgroundQuantitative reverse transcription PCR (qRT-PCR) is a robust and accessible method to assay gene expression and to infer gene regulation. Being a chain of procedures, this technique is subject to systematic error due to biological and technical limitations mainly set by the starting material and downstream procedures. Thus, rigorous data normalization is critical to grant reliability and repeatability of gene expression quantification by qRT-PCR. A number of ‘housekeeping genes’, involved in basic cellular functions, have been commonly used as internal controls for this normalization process. However, these genes could themselves be regulated and must therefore be tested a priori.MethodsWe evaluated eight potential reference genes for their stability as internal controls for RT-qPCR studies of olfactory gene expression in the antennae of Rhodnius prolixus, a Chagas disease vector. The set of genes included were: α-tubulin; β-actin; Glyceraldehyde-3-phosphate dehydrogenase; Eukaryotic initiation factor 1A; Glutathione-S-transferase; Serine protease; Succinate dehydrogenase; and Glucose-6-phosphate dehydrogenase. Five experimental conditions, including changes in age,developmental stage and feeding status were tested in both sexes.ResultsWe show that the evaluation of candidate reference genes is necessary for each combination of sex, tissue and physiological condition analyzed in order to avoid inconsistent results and conclusions. Although, Normfinder and geNorm software yielded different results between males and females, five genes (SDH, Tub, GAPDH, Act and G6PDH) appeared in the first positions in all rankings obtained. By using gene expression data of a single olfactory coreceptor gene as an example, we demonstrated the extent of changes expected using different internal standards.ConclusionsThis work underlines the need for a rigorous selection of internal standards to grant the reliability of normalization processes in qRT-PCR studies. Furthermore, we show that particular physiological or developmental conditions require independent evaluation of a diverse set of potential reference genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0862-x) contains supplementary material, which is available to authorized users.

Olfaction is fundamental for most animals and critical for different aspects of triatomine biology, including host-seeking, reproduction, avoidance of predators, and aggregation in shelters. Ethological and physiological aspects of these olfactory-mediated behaviors are well-understood, but their molecular bases are still largely unknown. Here we investigated changes in the molecular mechanisms at the peripheral olfactory level in response to different physiological and developmental conditions. For this, the antennal expression levels of the odorant (Orco) and ionotropic (IR8a, IR25a, and IR76b) coreceptor genes were determined in Rhodnius prolixus by means of quantitative real-time PCR (qRT-PCR) analysis. Gene expression changes were analyzed to test the effect of feeding and imaginal molt for both sexes. Moreover, we analyzed whether expression of these genes changed during the early life of adult bugs. Under these conditions bugs display distinct behavioral responses to diverse chemical stimuli. A significantly decreased expression was induced by blood feeding on all coreceptor genes. The expression of all genes was significantly increased following the imaginal molt. These results show that olfactory coreceptor genes have their expression altered as a response to physiological or developmental changes. Our study suggests that olfactory coreceptor genes confer adaptability to the peripheral olfactory function, probably underlying the known plasticity of triatomine olfactory-mediated behavior.

No abstract

No abstract

Systemic lupus erythematosus (SLE) is an autoimmune disease of the connective tissue with a large spectrum of clinical manifestations. Immune deregulation leads to autoantibody and immune complexes overproduction, complement activation, and persistent tissue inflammation. Considering that the current diagnosis depends on the interpretation of the complex criteria established by the American College of Rheumatology and that the disease course is characterized by unpredictable activations and remissions, each patient develops different manifestations, and therefore, the discovery of specific biomarkers is urgently required. Therefore, this study aimed to identify putative biomarkers for active and inactive SLE potentially capable in distinguishing laboratorial SLE from other autoimmune diseases. The 2D-DIGE proteomics technique was used to evaluate the differential abundance of proteins between patients with active SLE, inactive SLE, patients with other autoimmune disease, and healthy individuals. Six proteins showed increased abundance in active SLE (A) and inactive SLE (I) compared to the C and O groups, but not between groups A and I. There were two transthyretin (TTR) fragments or proteins with a structure similar to TTR (accession numbers: PDB: 1GKO_A and 2PAB_A), retinol-binding protein 4 (RBP4) isoform X1 (no information in databases such as UNIPROT), and antibody fragments. Two proteins, APO-AIV and SP-40,40, were upregulated in group A than in O and C and in group I versus C, but not in group I versus O. Therefore, we suggest these proteins to be considered as candidates for the diagnosis of SLE.