ArticleMolecular biology reports2025
Reference gene identification and evaluation for accurate qPCR normalization in Phenacoccus manihoti Matile-Ferrero (Hemiptera: Pseudococcidae).
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Abstract
backgroundThe Cassava mealybug (Phenacoccus manihoti), a major insect pest causing devastating yield losses in cassava, invaded India in 2020. Quantitative PCR (qPCR) is a widely employed molecular tool for gene expression profiling and crucial in functional genomics through RNAi. Many parameters can influence the robustness and reliability of qPCR results, so choosing an appropriate set of reference genes or housekeeping genes that yield consistent results across all experimental conditions is crucial for a successful qPCR assay. METHODS AND
resultsWe utilized four algorithms (geNorm, NormFinder, BestKeeper, and RefFinder) to assess the stability of six candidate reference genes across three experimental conditions: developmental stages, dietary sources, and dsRNA. For each experimental condition, three biological replicates were used, with three technical replicates for each sample. Across the developmental stages, EF2 and Actin exhibited the highest stability, whereas Actin and ATP5A1 were most stable across different dietary sources. ATP5A1 and β-tubulin were the most stable genes for dsRNA treatment studies. Normalization of the two target genes (Cht10, VgR) expression using the most stable and least stable reference genes demonstrated significant variations in expression levels.
conclusionIn the present study, ATP5A1 and Actin proved to be the most stable reference genes across all experimental conditions. This investigation represents the first comprehensive study of reference gene selection in P. manihoti, providing a crucial foundation for future functional gene expression studies in this insect.
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