Abstract
Reference genes have been utilized in estimating gene expression levels using quantitative reverse transcriptase-quantitative polymerase chain reaction (qRT-PCR) analysis. Aphidius gifuensis Ashmaed is one of the most widely used biological control agents for aphids. The biological properties of this species have been studied in detail, and current investigations are focused on elucidating the regulatory mechanisms in its host However, the appropriate reference genes for target gene expression studies have not been identified. In this study, the expression profiles of 12 candidate reference genes were evaluated under different experimental conditions(development stage, sex, tissue type, diet) by using dedicated algorithms, including geNorm, Normfinder, BestKeeper, and ΔCt. In addition, RefFinder was used to rank the overall stability of the candidate genes. Finally, we recommend three optimal reference genes for the normalization of qRT-PCR data in the presence of specific variables, which include ACTB, RPL13, and PPI for different developmental stages; RPS18, ACTB, and RPL13 for sexes; RPL13, PRII3, and RPS18 in different tissue types; and RPL13, RPL27, and ACTB in diverse diets. The present study has identified optimal reference genes that could be used in estimating the expression levels of specific genes under these conditions following the Minimum Information for publication of Quantitative real-time PCR Experiments (MIQE) guidelines, which would facilitate in advancements in functional genomics research on A. gifuensis.
Highlights
Real-time quantitative reverse transcriptase-polymerase chain reaction is the most sensitive and accurate method for determining mRNA expression levels of target genes under different experimental conditions and is commonly used to confirm the expression of relevant genes in high-throughput sequencing[1,2,3]
Twelve candidate reference genes were initially studied by reverse transcription polymerase chain reaction (RT-PCR)
The amplification efficiency (E) values of all candidate reference genes ranged from 88.96% to 96.20%, and the correlation coefficient (R2) values ranging from 0.971to 0.995 (Table 1 and S1 Fig),based on five-fold serial dilution of the pooled cDNA and the generated standard curve of each gene
Summary
Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) is the most sensitive and accurate method for determining mRNA expression levels of target genes under different experimental conditions and is commonly used to confirm the expression of relevant genes in high-throughput sequencing[1,2,3]. To accurately estimate gene expression levels, internal reference genes are used to normalize quantitative fluorescence data on the target gene. Numerous studies have shown that the expression levels of these housekeeping genes significantly vary under certain conditions and are not suitable as internal reference genes [11, 12]. QRT-PCR can be used as a rapid and reliable method for detecting and quantifying gene expression in different biological processes, some internal reference genes exhibit considerable differences in expression among various treatment settings [11]. The evaluation of housekeeping genes under different experimental conditions and the selection of the appropriate gene for normalization of expression is critical
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