Selection and Assessment of Reference Genes for Quantitative PCR Normalization in Migratory Locust Locusta migratoria (Orthoptera: Acrididae)

Abstract

Locusta migratoria is a classic hemimetamorphosis insect and has caused widespread economic damage to crops as a migratory pest. Researches on the expression pattern of functional genes in L. migratoria have drawn focus in recent years, especially with the release of genome information. Real-time quantitative PCR is the most reproducible and sensitive approach for detecting transcript expression levels of target genes, but optimal internal standards are key factors for its accuracy and reliability. Therefore, it's necessary to provide a systematic stability assessment of internal control for well-performed tests of target gene expression profile. In this study, twelve candidate genes (Ach, Act, Cht2, EF1α, RPL32, Hsp70, Tub, RP49, SDH, GAPDH, 18S, and His) were analyzed with four statistical methods: the delta Ct approach, geNorm, Bestkeeper and NormFinder. The results from these analyses aimed to choose the best suitable reference gene across different experimental situations for gene profile study in L. migratoria. The result demonstrated that for different developmental stages, EF1α, Hsp70 and RPL32 exhibited the most stable expression status for all samples; EF1α and RPL32 were selected as the best reference genes for studies involving embryo and larvae stages, while SDH and RP49 were identified for adult stage. The best-ranked reference genes across different tissues are RPL32, Hsp70 and RP49. For abiotic treatments, the most appropriate genes we identified were as follows: Act and SDH for larvae subjected to different insecticides; RPL32 and Ach for larvae exposed to different temperature treatments; and Act and Ach for larvae suffering from starvation. The present report should facilitate future researches on gene expression in L. migratoria with accessibly optimal reference genes under different experimental contexts.

Evaluation and Validation of Reference Genes for qRT-PCR Normalization in Frankliniella occidentalis (Thysanoptera:Thripidae)

“…Previous reference gene validation studies combined high and low temperature treatments, collectively referring to them as temperature treatment [18] , [27] , [40] , [41] . Interestingly, we found that the gene rankings for high and low temperature were different ( Fig.…”

Nuclear receptor hormone receptor 39 is required for locust moulting by regulating the chitinase and carboxypeptidase genes

“…The synthesis of cDNA and RT‐qPCR analysis were performed following previously described methods (Zhao et al ., ). Relative mRNA levels of target genes were calculated using the 2 −∆∆Ct method by normalizing to the expression of the internal marker gene ribosomal protein L32 , which exhibited the most stable expression at different stages and in different tissues (Livak and Schmittgen, ; Yang et al ., ). The primer information for each gene is listed in Table S2.…”

Selection of reference genes for quantitative real‐time polymerase chain reaction normalization inBradysia odoriphaga(Diptera: Sciaridae)

“…) and Locusta migratoria (Yang et al . ). α‐Tubulin , a type of cytoskeletal structure protein, is another commonly used reference gene.…”

“…In addition, our results show that Actin was the most stable reference gene in temperature experiments. It has been reported that, when suffering from different temperature treatments and starvation, Actin was selected as a stable reference gene in Myzus persicae (Kang et al 2017) and Locusta migratoria (Yang et al 2014). α-Tubulin, a type of cytoskeletal structure protein, is another commonly used reference gene.…”