Evaluation of reference genes for quantitative real-time PCR normalization in the scarab beetle Holotrichia oblita

Abstract

Quantitative real-time polymerase chain reaction (qPT-PCR) is commonly used to analyze gene expression, however, the accuracy of the normalized results is affected by the expression stability of reference genes. Holotrichia oblita (Coleoptera: Scarabaeidae) causes serious damage to crops. Reliable reference genes in H. oblita are needed for qRT-PCR analysis. Therefore, we evaluated 13 reference genes under biotic and abiotic conditions. RefFinder provided a comprehensive stability ranking, and geNorm suggested the optimal number of reference genes for normalization. RPL13a and RPL18 were the most suitable reference genes for developmental stages, tissues, and temperature treatments; RPL13a and RPS3 were the most suitable for pesticide and photoperiod treatments; RPS18 and RPL18 were the most suitable for the two sexes. We validated the normalized results using odorant-binding protein genes as target genes in different tissues. Compared with the selected suitable reference genes, the expression of OBP1 in antennae, abdomen, and wings, and OBP2 in antennae and wings were overestimated due to the instability of ACTb. These results identified several reliable reference genes in H. oblita for normalization, and are valuable for future molecular studies.

Evaluation of suitable reference genes for expression analysis using quantitative real‐time polymerase chain reaction in the parasitoid Exorista sorbillans (Diptera: Tachinidae)

“…Nine commonly used reference genes in insects were selected from transcriptome data set of E. sorbillans (Qu et al, 2018; Xie et al, 2020). Gene symbols, accession numbers, primers, amplicon length, amplification efficiency and the linear correlation coefficient ( R 2 ) are listed in Table 1.…”

Selection and evaluation of RT-qPCR reference genes for expression analysis in the tiny egg parasitoid wasp, Trichogramma dendrolimi matsumura (Hymenoptera: Trichogrammatidae)

“…for normalizing the expression level of target genes in Hymenopteran insects and other insects, such as A. mellifera (RP49, RPL13a, and RPS18) [34], L. japonica (RPL18, RPL27, and RPS18) [20], Solenopsis invicta (RPL18) [48], Tetranychus cinnabarinus (RPL13a, and RPS18) [22], and Holotrichia oblita (RPL13a, RPL18, RPS18, RPS3, and RPS6) [49].…”

Selection and evaluation of qPCR reference genes for expression analysis in the tiny egg parasitoid wasp,Trichogramma dendrolimiMatsumura (Hymenoptera: Trichogrammatidae)

“…The gene family of ribosomal proteins is the component of ribosomes and is constantly expressed throughout all developmental stages and in all living cells of the organisms [25]. Some of the genes have been used for normalizing the expression level of target genes in Hymenopteran insects and other insects, such as A. mellifera ( RP49, RPL13a , and RPS18 ) [34], L. japonica ( RPL18, RPL27 , and RPS18 ) [20], Solenopsis invicta ( RPL18 ) [48], Tetranychus cinnabarinus ( RPL13a , and RPS18 ) [22], and Holotrichia oblita ( RPL13a, RPL18, RPS18, RPS3 , and RPS6 ) [49].…”