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

BackgroundRhodnius 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.ResultsThe 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.ConclusionsThe 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.

Identification and Validation of Reference Genes for Quantitative Real-Time PCR in Drosophila suzukii (Diptera: Drosophilidae)

“…Previously, Actin has been considered to be one of the best reference genes for assessing gene expression in many insects [29], [30], and GAPDH was also frequently used as a reference gene [11], [31]. However, they were not the best reference genes in the present analysis and several studies have demonstrated that the stability of GAPDH expression was low in certain conditions [32].These results further suggest that the expression stability of reference genes is affected by different experimental conditions.…”

Selection and evaluation of reference genes for quantitative gene expression studies in cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

“…This gene was also reported to be the most stably expressed gene in larvae of the oriental leafworm moth Spodoptera litura under temperature stress and across different developmental stages (Lu et al, 2013) and in the head of the bee Apis mellifera infected with bacteria (Scharlaken et al, 2008). However, some other studies have reported reduced expression stability of this gene like in salivary glands of kissing-bug Rhodnius prolixus when infected with Protozoa Trypanosoma cruzi, in labial glands and fat body of the bumblebees Bombus lucorum and Bombus terrestris and across different life stages of the spider mite Tetranychus cinnabarinus (Hornakova et al, 2010;Paim et al, 2012;Sun et al, 2010).…”

Identification and Validation of Reference Genes for Gene Expression Analysis Using Quantitative PCR in Spodoptera litura (Lepidoptera: Noctuidae)

“…Scharlaken [26] also identified GAPDH as the most stable gene in the head of the honeybee after bacterial challenge. However, several studies have demonstrated that the stability of GAPDH expression was low in certain conditions, such as certain life stages of Tetranychus cinnabarinus , in the labial gland and fat body in Bombus terrestris and Bombus lucorum , and in the salivary glands after Trypanosoma cruzi injection [17], [18], [25]. These results further suggest that the expression stability of reference genes is affected by different experimental conditions.…”

“…Based on the described insect reference genes in literature, the NCBI database (http://www.ncbi.nlm.nih.gov) was searched for available S. litura sequences: ACTB [17], UCCR , FTZF1 , GAPDH [6], EF1 [2], [3] and AK [18]. We explored whether UCCR and FTZF1 could be used as reference genes.…”