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

Abstract

Selecting stable reference genes for reverse transcription quantitative real‐time polymerase chain reaction analysis is critically important for gene expression. Bradysia odoriphaga is the most serious pest of Chinese chives, but our knowledge of its genetics is limited. In the present study, five housekeeping genes (β‐Actin, α‐Tubulin, RPS7, GAPDH and 18S rRNA) were cloned from B. odoriphaga using the combined techniques of reverse transcription polymerase chain reaction with rapid amplification of cDNA ends. The five genes, together with RPS15, were quantified for transcription stability in B. odoriphaga under various experimental conditions. Their expression stability was evaluated using four different algorithms (a comparative ΔCt method, GeNorm, NormFinder and BestKeeper). Additionally, we used an online Web‐based tool (RefFinder) to assign an overall final rank to each candidate gene. These analyses identified 18S, RPS15 and RPS7 as the most stable reference genes across developmental stages. 18S, RPS7 and Tubulin were the most stable reference genes for monitoring gene expression across different tissues of adults. RPS7, GAPDH and Tubulin were selected as the best reference genes across different larval tissues. GAPDH and RPS15 were selected to normalize gene expression for experiments of insecticide treatments. Actin and GAPDH are considered suitable reference genes in experiments of temperature treatments. Actin and Tubulin are considered suitable reference genes for starvation experiments. This study provides an important supplement of suitable reference genes for B. odoriphaga and these results provide clues toward the understanding of the genes’ biological functions in B. odoriphaga.

Involvement of Chemosensory Protein BodoCSP1 in Perception of Host Plant Volatiles in Bradysia odoriphaga

“…The relative expression levels of BodoCSP1 were analyzed using the 2 –ΔΔ CT method . Different reference gene combinations were used to normalize the expression of the target genes, 18S , Tubulin , and RPS7 for various tissues of adults, 18S , RPS15 , and RPS7 for different developmental stages . Each assay was conducted in three technical replicates using three biological samples.…”

Selection and Validation of Reference Genes for RT-qPCR Normalization in Bradysia odoriphaga (Diptera: Sciaridae) Under Insecticides Stress

“…On the other hand, the difference in ranking of the reference genes under the stress of different insecticides in this study demonstrates the importance of evaluating their use under different sets of insecticides. Our findings provide more comprehensive information regarding reference genes selection under insecticide stress compared to the previous studies on B. odoriphaga (Shi et al, 2016;Tang et al, 2019).…”

“…In this case, it’s important to choose reliable reference genes for gene expression analysis under various experimental conditions. Though appropriate references genes have been identified in B. odoriphaga under different biotic and abiotic conditions ( Shi et al, 2016 ; Tang et al, 2019 ), a piece of comprehensive information is lacking for B. odoriphaga stressed by different groups of insecticides. Therefore, in this study, nine commonly used reference genes elongation factor 1α (EF-1α) , actin2 (ACT) , elongation factor 2α (EF-2α) , glucose-6-phosphate dehydrogenase (G6PDH) , glyceraldehyde-3-phosphate dehydrogenase (GAPDH) , ribosomal protein L10 (RPL10) , ribosomal protein S3 (RPS3) , ubiquitin-conjugating enzyme (UBC) , and α-tubulin (TUB) were analyzed to assess their suitability for normalizing RT-qPCR data for B. odoriphaga under the stress of insecticides (phoxim, chlorpyrifos, imidacloprid, and chlorfluazuron).…”

Functional Characterization of an α-Esterase Gene Associated with Malathion Detoxification in Bradysia odoriphaga

“…The results were determined using the 2 −ΔΔCT method (Livak and Schmittgen, 2001) 24 and normalized using different reference genes. 25 18S, RPS15, and RPS7 for different developmental stages, 18S and RPS15 for various tissues of larvae, and RPL18 and RPS15 for samples from insecticide-treated insects. Each assay was performed with three biological replicates and three technical replicates.…”