Evaluation of reference genes at different developmental stages for quantitative real-time PCR in Aedes aegypti

Abstract

The mosquito Aedes aegypti (Ae. aegypti) is the most notorious vector of illness-causing viruses such as Dengue, Chikugunya, and Zika. Although numerous genetic expression studies utilizing quantitative real-time PCR (qPCR) have been conducted with regards to Ae. aegypti, a panel of genes to be used suitably as references for the purpose of expression-level normalization within this epidemiologically important insect is presently lacking. Here, the usability of seven widely-utilized reference genes i.e. actin (ACT), eukaryotic elongation factor 1 alpha (eEF1α), alpha tubulin (α-tubulin), ribosomal proteins L8, L32 and S17 (RPL8, RPL32 and RPS17), and glyceraldeyde 3-phosphate dehydrogenase (GAPDH) were investigated. Expression patterns of the reference genes were observed in sixteen pre-determined developmental stages and in cell culture. Gene stability was inferred from qPCR data through three freely available algorithms i.e. BestKeeper, geNorm, and NormFinder. The consensus rankings generated from stability values provided by these programs suggest a combination of at least two genes for normalization. ACT and RPS17 are the most dependably expressed reference genes and therefore, we propose an ACT/RPS17 combination for normalization in all Ae. aegypti derived samples. GAPDH performed least desirably, and is thus not a recommended reference gene. This study emphasizes the importance of validating reference genes in Ae. aegypti for qPCR based research.

Figure 1. Box-whisker plots depicting expression levels in terms of Ct values for the seven candidate genes in ten different samples, and across all sample types.

Boxes encompass 25th to 75th percentiles. Whisker caps denote maximum and minimum Ct values.

Figure 2

(A) Average stability values (M) of genes in individual developmental stages and cell culture. (B) Pairwise variation (V) analysis of candidate reference genes. For each Figure the graphs represent (A) 0 to 3 hour embryos (B) 3 to 6 hour embryos (C) 6 to 9 hour embryos (D) 9 to 12 hour embryos (E) 12 to 18 hour embryos (F) 18 to 24 hour embryos (G) 24 to 48 hour embryos (H) 48 to 72 hour embryos (I) First instar larvae (J) Second instar larvae (K) Third instar larvae (L) Fourth instar larvae (M) Pupae (N) Adult Male (O) Adult Female (P) Adult Female, 6 hours Post-Blood Meal (Q) Aag2 cells.