Abstract
Quantitative reverse transcription PCR (RT-qPCR) is one of the most efficient, reliable and widely used techniques to quantify gene expression. In this study, we evaluated the performance of six southern corn rootworm, Diabrotica undecimpunctata howardi (Barber), housekeeping genes (HKG), β-actin (Actin), β-tubulin (Tubulin), elongation factor 1 alpha (EF1α), glyceraldehyde-3 phosphate dehydrogenase (GAPDH), 40 S ribosomal protein S9 (RpS9) and ubiquitin-conjugating protein (Ubi), under different experimental conditions such as developmental stage, exposure of neonate and adults to dsRNA, exposure of adults to different temperatures, different 3rd instar larva tissues, and neonate starvation. The HKGs were analyzed with four algorithms, including geNorm, NormFinder, BestKeeper, and delta-CT. Although the six HKGs showed a relatively stable expression pattern among different treatments, some variability was observed. Among the six genes, EF1α exhibited the lowest Ct values for all treatments while Ubi exhibited the highest. Among life stages and across treatments, Ubi exhibited the least stable expression pattern. GAPDH, Actin, and EF1α were among the most stable HKGs in the majority of the treatments. This research provides HKG for accurate normalization of RT-qPCR data in the southern corn rootworm. Furthermore, this information can contribute to future genomic and functional genomic research in Diabrotica species.
Highlights
The southern corn rootworm (SCR), Diabrotica undecimpunctata howardi (Barber), is a polyphagous/omnivorous plant pest found in the United States east of the Rocky Mountains, southern Canada, and northern Mexico[1,2]
It is well known that the sensitivity, specificity and accuracy of RNA extraction and quantitative real-Time PCR (RT-qPCR) depend on several factors, including primer efficiency, the number of replications, but most importantly the choice of appropriate reference genes[16,17]
RT-qPCR products generated with each set of primers were confirmed by the presence of a single peak in melting curve analyses
Summary
Function Cell mortality, structure, and Integrity[34,35] Component of the 40 S subunit of ribosome[34]. The selected genes are among the six most commonly reported genes in the literature[15,17]: Actin[34,35], Tubulin[34], EF1α34, GAPDH34,36, 40 S ribosomal protein S9 (RpS9)[34] and ubiquitin-conjugating protein (Ubi)[17]. These HKGs were tested across different larval tissues (head, midgut, and carcass [integument + fat body]) and developmental stages (egg, first, second and third instar larvae, pupa, adult male and female). SCR neonates and adults were exposed to different stressors, including ingested Snf[7] dsRNA, starvation, and a range of temperatures (8 °C, 24 °C and 36 °C)
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