Assessment of suitable reference genes for qRT-PCR analysis in Adelphocoris suturalis

Abstract

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the most commonly-used tool for measurement of gene expression, but its accuracy and reliability depend on appropriate data normalization with the use of one or more stable reference genes. Adelphocoris suturalis is one of the most destructive pests of cotton, but until recently knowledge of its underlying molecular physiology had been hindered by a lack of molecular resources. To facilitate research on this pest, we evaluated 12 common housekeeping genes studied in insects (GAPDH, ACT, βACT, TBP, SDH, βTUB, EF1γ, EF1α, EF1δ, RPL32, RPS15, and RPL27) for their expression stability in A. suturalis when subjected to various experimental treatments, including three biotic (developmental stage and sex, tissue type, and metathoracic scent gland for varying developmental stages and sexes) and one abiotic (RNA interference injection) conditions. Four dedicated algorithms (ΔCt method, geNorm, BestKeeper and NormFinder) were used to analyze gene expression stability. In addition, RefFinder provided an overall ranking of the stability/suitability of these candidates. This study is the first to provide a comprehensive list of suitable reference genes for gene expression analyses in A. suturalis, which can serve to facilitate transcript expression study of related biological processes in this and related species.

Keywords

• Adelphocoris suturalis
• reference gene
• qRT-PCR
• normalization
• expression stability