Abstract
Finding a suitable reference gene is the key for qRT-PCR analysis. However, none of the reference gene discovered thus far can be utilized universally under various biotic and abiotic experimental conditions. In this study, we further examine the stability of candidate reference genes under a single abiotic factor, insecticide treatment. After being exposed to eight commercially available insecticides, which belong to five different classes, the expression profiles of eight housekeeping genes in the sweetpotato whitefly, Bemisia tabaci, one of the most invasive and destructive pests in the world, were investigated using qRT-PCR analysis. In summary, elongation factor 1α (EF1α), α-tubulin (TUB1α) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were identified as the most stable reference genes under the insecticide treatment. The initial assessment of candidate reference genes was further validated with the expression of two target genes, a P450 (Cyp6cm1) and a glutathione S-transferase (GST). However, ranking of reference genes varied substantially among intra- and inter-classes of insecticides. These combined data strongly suggested the necessity of conducting custom reference gene selection designed for each and every experimental condition, even when examining the same abiotic or biotic factor.
Highlights
Quantitative real-time reverse transcription polymerase chain reaction is one of the most effective and sensitive techniques for gene expression analysis [1,2,3,4]
Reliable reference genes for gene expression analysis based on different experimental conditions should be selected
A set of reliable reference genes for gene expression analysis in the B. tabaci biotype Q, one of the most invasive and destructive pest in the world, after exposure to eight commonly used insecticides was selected and valuated with two target genes, a P450 gene (Cyp6cm1) and glutathione S-transferase gene (GST)
Summary
Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) is one of the most effective and sensitive techniques for gene expression analysis [1,2,3,4]. Enabling comparisons across different samples, qRT-PCR data must be normalized to correct variations in pipetting, RNA concentration, reverse-transcription, and efficiency of PCR amplification [2,5,6]. It is not surprising that no single universal reference is available for four different lepidopteran insect species [24]. In this context, reliable reference genes for gene expression analysis based on different experimental conditions should be selected
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