Comparison of the differential proteins expression level in two groups.

<p>CK: the control, samples treated with cotton balls soaked distilled water only. T: the test, samples treated with cotton balls soaked 0.8% (8 mg/mL) colchicine solution.</p

Obs. pI and Mr, observed pI, Mr calculated from the 2D-gels with PDQuest 8.0.1 software according to standard marker proteins.

<p>ER: endoplasmic reticulum, PM: Plasma Membrane, M: Mitochondrion.</p

Functional categorization of different proteins. Digitals stand for the protein number of each functional category.

<p>Functional categorization of different proteins. Digitals stand for the protein number of each functional category.</p

Representative 2-DE gels of untreated (A) and colchicine treated (B) microsporangia.

<p>Twenty-seven of the spots showing at least a 2-fold change with <i>p</i><0.05 were analyzed by MALDI-TOF/TOF-MS.</p

Reference Gene Selection for qRT-PCR Analysis in the Sweetpotato Whitefly, <em>Bemisia tabaci</em> (Hemiptera: Aleyrodidae)

<div><h3>Background</h3><p>Accurate evaluation of gene expression requires normalization relative to the expression of reliable reference genes. Expression levels of “classical” reference genes can differ, however, across experimental conditions. Although quantitative real-time PCR (qRT-PCR) has been used extensively to decipher gene function in the sweetpotato whitefly <em>Bemisia tabaci</em>, a world-wide pest in many agricultural systems, the stability of its reference genes has rarely been validated.</p> <h3>Results</h3><p>In this study, 15 candidate reference genes from <em>B. tabaci</em> were evaluated using two Excel-based algorithms <em>geNorm</em> and <em>Normfinder</em> under a diverse set of biotic and abiotic conditions. At least two reference genes were selected to normalize gene expressions in <em>B. tabaci</em> under experimental conditions. Specifically, for biotic conditions including host plant, acquisition of a plant virus, developmental stage, tissue (body region of the adult), and whitefly biotype, <em>ribosomal protein L29</em> was the most stable reference gene. In contrast, the expression of <em>elongation factor 1 alpha</em>, <em>peptidylprolyl isomerase A</em>, <em>NADH dehydrogenase</em>, <em>succinate dehydrogenase complex subunit A</em> and <em>heat shock protein 40</em> were consistently stable across various abiotic conditions including photoperiod, temperature, and insecticide susceptibility.</p> <h3>Conclusion</h3><p>Our finding is the first step toward establishing a standardized quantitative real-time PCR procedure following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guideline in an agriculturally important insect pest, and provides a solid foundation for future RNA interference based functional study in <em>B. tabaci</em>.</p> </div

Reference genes selected by <i>geNorm</i> under various biotic conditions.

<p>The expression stability measure (<i>M</i>) is the mean of the stability values of the remaining genes. The least stable genes have the highest <i>M</i> values. The genes listed here are considered stable based on a cutoff <i>M</i> value of less than 0.5. Each circle with a distinct color represents a different set of biotic condition. Genes located within one circle are stable under a specific biotic condition, and genes shared with multiple circles are stable across those conditions.</p

Ranking of candidate reference genes in response to abiotic factors.

“1”<p>: Thiamethoxam-resistant and -susceptible whiteflies.</p>“2”<p>: <u>S</u>tability <u>V</u>alue was evaluated by <i>Normfinder</i>.</p

Primers used for qRT-PCR analysis.

“1”<p>: F, forward primer; R, reverse primer;</p>“2”<p>: Tm, Annealing temperature;</p>“3”<p>: E, Efficiency;</p>“4”<p>: <i>R</i>², Coefficient of determination.</p

Reference genes selected by <i>geNorm</i> under various abiotic conditions.

<p>The expression stability measure (<i>M</i>) is the mean of the stability values of the remaining genes. The least stable genes have the highest <i>M</i> values. The genes listed here are considered stable based on a cutoff <i>M</i> value of less than 0.5. Each circle with a distinct color represents a different set of biotic condition. Genes located within one circle are stable under a specific abiotic condition, and genes shared with multiple circles are stable across those conditions.</p

Recommended reference genes for various experimental conditions.

<p>Recommended reference genes for various experimental conditions.</p