Selection and Validation of Reference Genes For qRT-PCR Analysis of Rhopalosiphum padi (Hemiptera: Aphididae)

Abstract

Rhopalosiphum padi (L.) (Hemiptera: Aphididae) is an important cosmopolitan pest in cereal crops. Reference genes can significantly affect qRT-PCR results. Therefore, selecting appropriate reference genes is a key prerequisite for qRT-PCR analyses. This study was conducted to identify suitable qRT-PCR reference genes in R. padi. We systematically analyzed the expression profiles of 11 commonly used reference genes. The ΔCt method, the BestKeeper, NormFinder, geNorm algorithms, and the RefFinder online tool were used to evaluate the suitability of these genes under diverse experimental conditions. The data indicated that the most appropriate sets of reference genes were β-actin and GAPDH (for developmental stages), AK and TATA (for populations), RPS18 and RPL13 (for tissues), TATA and GAPDH (for wing dimorphism), EF-1α and RPS6 (for antibiotic treatments), GAPDH and β-actin (for insecticide treatments), GAPDH, TATA, RPS18 (for starvation-induced stress), TATA, RPS6, and AK (for temperatures), and TATA and GAPDH (for all conditions). Our study findings, which revealed the reference genes suitable for various experimental conditions, will facilitate the standardization of qRT-PCR programs, while also improving the accuracy of qRT-PCR analyses, with implications for future research on R. padi gene functions.

Selection and validation of reference genes for RT-qPCR-based analyses of Anastatus japonicus Ashmead (Hymenoptera: Helicopteridae)

“…However, the absence of poly-A in rRNA makes it impossible to isolate it using oligo (dT)-based techniques, which is a significant disadvantage when employing rRNA as a normalizer in RT-qPCR experiments ( Xue et al, 2010 ). Thus, we focused on genes transcribed by RNA polymerase II whose transcripts can be isolated using oligo (dT), like the genes linked to GAPDH, ACTIN and ribosomal proteins (e.g., RPS3a, RPS6, and RPL13) ( Galetto et al, 2013 ; Xu et al, 2021 ; Li et al, 2021 ; Shen et al, 2022 ). And the TATA gene is required for initiating the RNA polymerase (I, II, and III)-mediated transcription of genes with promoters with or without a TATA box ( Pugh, 2000 ).…”

Identifying optimal reference genes for gene expression studies in Eurasian spruce bark beetle, Ips typographus (Coleoptera: Curculionidae: Scolytinae)

“…RPS13 and RPS7 exhibited the most stable expression under larval-crowding conditions in Mythimna separata 63 . Similarly, different developmental stages ( RPL32, RPS18 ) and different tissues ( RPS18 ) of Spodoptera frugiperda showed the most stable expression 64 , whereas RPS26 and RPL32 genes showed the same in Thermobia domestica 65 ; RPS11 , RPL28 , and RPL10 genes showed Tuta absoluta 58 ; RPS18 and RPL13 genes showed Rhopalosiphum padi tissues 66 . Sellamuthu et al 43 reported that RPS3 is stably expressed in the developmental stage, sex-specific and tissue-specific conditions in pine-feeding bark beetle, I. sexdentatus .…”

Reference Gene Selection for Normalizing Gene Expression in Ips Sexdentatus (Coleoptera: Curculionidae: Scolytinae) Under Different Experimental Conditions

“…Similarly, RPL9 and RPL10 genes showed higher expression stability in different developmental stages and tissues of Sogatella furcifera (An et al, 2016 ), whereas RPS26 and RPL32 genes showed the same in Thermobia domestica (Bai et al, 2021 ). Furthermore, RPS18 and RPL13 genes showed the highest expression stability in Rhopalosiphum padi tissues (Li et al, 2021 ). Wang et al ( 2014 ) reported RPL22e as the most stable reference gene comparing male and female Mylabris cichorii .…”