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Selection and Evaluation of Reference Genes for qRT-PCR in Spodoptera frugiperda (Lepidoptera: Noctuidae).

ABSTRACT: As an accurate and convenient technique, the qRT-PCR is always used in the quantitative expression analysis of functional genes. Normalization of the data relies on stable reference genes. The fall armyworm Spodoptera frugiperda (J. E. Smith) is an important invasive and migratory pest that seriously threatens corn production around the world. In this paper, we selected 10 candidate reference genes (18S, AK, RPL10, RPS24, 28S, SOD, ATP, GAPDH, ACT, and a-TUB) and determined their expression levels under different conditions (different developmental stages, various tissues, mating status, hormones, diets, and temperatures). Subsequently, the stability of reference genes was evaluated by four algorithms (Delta Ct method, geNorm, NormFinder, BestKeeper). The optimal combination of reference genes for each treatment was obtained by geNorm. Finally, the comprehensive ranks were determined by the online tool RefFinder. Results showed that the most stable reference genes were SOD, RPL10, and RPS24 for developmental stages, α-TUB, RPL10, and ATP for different tissues, AK, RPL10, and 18S for mating status, 18S and AK under hormone treatment, 18S, RPL10, and SOD under diet treatment, RPL10, 18S, and RPS24 under temperature treatment. This study confirmed recent data on a few reference genes and provided an evaluation of a number of additional reference genes of S. frugiperda under various conditions.

SUBMITTER: Han S

PROVIDER: S-EPMC8538597 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

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Selection and Evaluation of Reference Genes for qRT-PCR in Spodoptera frugiperda (Lepidoptera: Noctuidae).

Han Shipeng S Qin Qiuju Q Wang Da D Zhou Yayuan Y He Yunzhuan Y

Insects 20211003 10

As an accurate and convenient technique, the qRT-PCR is always used in the quantitative expression analysis of functional genes. Normalization of the data relies on stable reference genes. The fall armyworm Spodoptera frugiperda (J. E. Smith) is an important invasive and migratory pest that seriously threatens corn production around the world. In this paper, we selected 10 candidate reference genes (18S, AK, RPL10, RPS24, 28S, SOD, ATP, ...[more]

PMID: 34680671

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Project description:Quantitative real-time PCR (qRT-PCR) is a reliable and reproducible technique for measuring and evaluating changes in gene expression. The most common method for analyzing qRT-PCR data is to normalize mRNA levels of target genes to internal reference genes. Evaluating and selecting stable reference genes on a case-by-case basis is critical. The present study aimed to facilitate gene expression studies by identifying the most suitable reference genes for normalization of mRNA expression in qRT-PCR analysis of the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae). For this purpose, three software tools (geNorm, NormFinder and BestKeeper) were used to investigate 10 candidate reference genes in nine developmental stages and five different tissues (epidermis, head, midgut, fat body and hemolymph) in three larval physiological stages (molting, feeding and wandering stages) of, S. exigua. With the exception of 18S ribosomal RNA (18S), all other candidate genes evaluated, β-actin1(ACT1), β-actin2 (ACT2), elongation factor1(EF1), elongation factor 2 (EF2), Glyceralde hyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (L10), ribosomal protein L17A (L17A), superoxide dismutase (SOD), α-tubulin (TUB),proved to be acceptable reference genes. However, their suitability partly differed between physiological stages and different tissues. L10, EF2 and L17A ranked highest in all tissue sample sets. SOD, ACT2, GAPDH, EF1 and ACT1 were stably expressed in all developmental stage sample sets; ACT2, ACT1 and L10 for larvae sample sets; GAPDH, ACT1 and ACT2 for pupae and adults; SOD and L17A for males; and EF2 and SOD for females. The expression stability of genes varied in different conditions. The findings provided here demonstrated, with a few exceptions, the suitability of most of the 10 reference genes tested in tissues and life developmental stages. Overall, this study emphasizes the importance of validating reference genes for qRT-PCR analysis in S. exigua.

Project description:The aim of this experiment was to compare the transciptome of the fall armyworm (Spodoptera frugiperda) strain SUS (a laboratory insecticide-susceptible standard) with organophosphate (OP) and pyrethroid (PYR) resistant strains were collected in cornfields located in Minas Gerais and Mato Grosso States, Brazil, in 2008 and maintained in the laboratory under selection with chlorpyrifos (at increasing discriminating doses from 100 up to 400 M-BM-5g of insecticide/g of insect in a topical bioassay) or lambda-cyhalothrin (from 8.4 up to 27 M-BM-5g of insecticide/g of insect) respectively The custom microarray used in this study was designed using the Agilent eArray platform (Agilent Technologies). A SurePrint HD (8M-CM-^W15k) expression array was designed using the base composition and the best probe methodologies to design sense orientation 60-mer probes with a 3M-bM-^@M-2 bias. The FAW EST database (SPODOBASE) was used as the reference transcriptome (Negre, Hotelier et al. 2006). These sequences are derived from 8 cDNA libraries as follows: Sf1F: Fat body, Sf1H: Hemocyte, Sf1M: Midgut, Sf1P: Pool of various tissues, Sf2H: Immune Challenged hemocytes, Sf2L: Sf21 Cell lines sequences, Sf2M: Xenobiotic Induced Midgut and Sf9L: Sf9 cell lines sequences. All assembled contigs and singlets were kindly sent by the website maintainers. The BLAST2GO software v.2.3.1 (http://www.blast2go.org) was used to annotate the EST database. 60-mer probes were designed for all 7,552 assembled contigs and 5,519 annotated singlets (BlastX), totaling 13,071 sequences. For contigs encoding detoxification enzymes (P450s, GSTs and CEs) three probes were designed. Additional probe groups for 15 control genes were also included. For reference all sequences are included in the zip file with array data. References: Negre, V., T. Hotelier, et al. (2006). "SPODOBASE : an EST database for the lepidopteran crop pest Spodoptera." BMC Bioinformatics 7: 322. Two-condition experiment. Slide 1: SUS vs. OP S. fugiperda strains. Slide 2: SUS vs. PYR S. fugiperda strains. Biological replicates: 4 pools of RNA extracted from four pools of 5 second instar larvae. Technical Replicates: None, the biological replicates incorporated a dye swap. Total replication: four replicates for each strain.

Dataset Information

Selection and Evaluation of Reference Genes for qRT-PCR in Spodoptera frugiperda (Lepidoptera: Noctuidae).

Publications

Selection and Evaluation of Reference Genes for qRT-PCR in <i>Spodoptera frugiperda</i> (Lepidoptera: Noctuidae).

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