Looking for reference genes for real‐time quantitative PCR experiments in Rhodnius prolixus (Hemiptera: Reduviidae)

Abstract

Quantitative real-time PCR (qPCR) has become one of the most used techniques to measure gene expression. However, normalization of gene expression data against reference genes is essential, although these are usually used without any kind of validation. The expression of seven genes was compared in organs of Rhodnius prolixus under diverse conditions, using published software to test gene expression stability. Rp18S and elongation factor 1 (RpEF -1) were the most reliable genes for normalization in qPCR when gene expression in different organs was compared. Moreover, both genes were found to be the best references when transcript levels were compared in the posterior midgut of insects infected with Trypanosoma cruzi. Rp18S was also the best reference gene in the fat bodies of unfed and fed insects. By contrast, RpEF-1 was found to be the best reference gene for comparison between posterior midguts, and RpMIP or RpActin should be used to compare gene expression in the ovaries. Although Rp18S is indicated here as the best reference in most cases, reports from the literature show that it is difficult to find an optimum reference gene. Nevertheless, validation of candidate genes to be taken as references is important when new experimental conditions are tested to avoid incorrect data interpretation.

Functional Characterization of Odorant Binding Protein 27 (RproOBP27) From Rhodnius prolixus Antennae

“…PCR studies were performed using GoTaq ® Green Master Mix kit (Promega, Madison, WI, United States). R. prolixus ’ ribosomal gene 18S ( RproR18S ) was used as the reference gene (Majerowicz et al, 2011). PCRs were performed on Veriti ® Thermal Cycler-96 well thermocycler (Applied Biosystems, Foster City, CA, United States), consisting of 35 cycles for RproOBPs and 25 cycles for RproR18S under the following conditions, 94°C for 3 min, followed by denaturation steps at 94°C for 30 s, annealing temperature was set according to each primer pair ( Supplementary Table S1 ) for 30 s and the extension step at 72°C for 1 min and 30 s, finally followed by 72°C for 10 min.…”

Vaccination with cyclin-dependent kinase tick antigen confers protection against Ixodes infestation

“…Serial dilutions of the cDNA were used to construct a calibration curve, and reaction efficiencies between 85 and 100% were determined from calibration curves for each set of primers in 10-L reactions. The relative expression ratio of CDK genes in each experiment was calculated according to the model described by Pfaffl (2001) in the Relative Expression Software Tool (REST-MCS , version 2) (Pfaffl, 2001), and normalized with tick Elongation Factor gene (ELF) (Majerowicz et al, 2011). The relative expression of the calibrators was assigned a value of 1 unit.…”

Genomic and functional characterization of a methoprene-tolerant gene in the kissing-bug Rhodnius prolixus

“…Results were normalized using b-actin and ribosome S18 as reference genes (see Supplemental Fig. 3) (Majerowicz et al, 2011). Primer sequences for RT-qPCR analysis were: b-actin fwd, 5 0 -CACCCCAGCAATGTATGTAG-3 0 ; b-actin rev, 5 0 -ACCATCAGGAAGTTCGTAAG-3 0 ; S18 fwd, 5 0 -TCCTTCGTGCTAGG AATTGG-3 0 ; S18 rev, 5 0 -GTACAAAGGGCAGGGACGTA-3 0 ; 109 bp of the exon 3 of the Rp-Met isoform 1 (JN416985) ( Table 1).…”