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Validation of reference housekeeping genes for gene expression studies in western corn rootworm (Diabrotica virgifera virgifera).

Rodrigues TB, Barros Rodrigues T, Khajuria C, Wang H, Matz N, Cunha Cardoso D, Valicente FH, Zhou X, Siegfried B - PLoS ONE (2014)

Bottom Line: In general, normalization of results using a highly stable housekeeping gene (HKG) as an internal control is recommended and necessary.EF1a was also the most stable for Bt exposure and among different tissues.These results will enable researchers to use more accurate and reliable normalization of qRT-PCR data in WCR experiments.

View Article: PubMed Central - PubMed

Affiliation: CAPES Foundation, Ministry of Education of Brazil, Brasília, Federal District, Brazil, and Federal University of Lavras, Lavras, Minas Gerais, Brazil.

ABSTRACT
Quantitative Real-time PCR (qRT-PCR) is a powerful technique to investigate comparative gene expression. In general, normalization of results using a highly stable housekeeping gene (HKG) as an internal control is recommended and necessary. However, there are several reports suggesting that regulation of some HKGs is affected by different conditions. The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a serious pest of corn in the United States and Europe. The expression profile of target genes related to insecticide exposure, resistance, and RNA interference has become an important experimental technique for study of western corn rootworms; however, lack of information on reliable HKGs under different conditions makes the interpretation of qRT-PCR results difficult. In this study, four distinct algorithms (Genorm, NormFinder, BestKeeper and delta-CT) and five candidate HKGs to genes of reference (β-actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; β-tubulin; RPS9, ribosomal protein S9; EF1a, elongation factor-1α) were evaluated to determine the most reliable HKG under different experimental conditions including exposure to dsRNA and Bt toxins and among different tissues and developmental stages. Although all the HKGs tested exhibited relatively stable expression among the different treatments, some differences were noted. Among the five candidate reference genes evaluated, β-actin exhibited highly stable expression among different life stages. RPS9 exhibited the most similar pattern of expression among dsRNA treatments, and both experiments indicated that EF1a was the second most stable gene. EF1a was also the most stable for Bt exposure and among different tissues. These results will enable researchers to use more accurate and reliable normalization of qRT-PCR data in WCR experiments.

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Related in: MedlinePlus

Ct values of the five candidate reference genes in all four experiments.Black bars indicate an average between maximum (Max) and minimum (Min) Ct values.
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pone-0109825-g001: Ct values of the five candidate reference genes in all four experiments.Black bars indicate an average between maximum (Max) and minimum (Min) Ct values.

Mentions: The expression profiles of all qRT-PCR products for all primers and all experiments are shown in Figure 1. The expression levels of all HKGs were measured by the Ct value, which is the number of PCR cycles needed to reach a specific threshold level of detection and is inversely correlated with the quantity of initial RNA template. For qRT-PCR normalization, a moderately expressed reference gene is preferred because extremely high or low expression of a HKG could introduce variability to data analysis [31], so a standard Ct value range was analyzed for all four experiments (Fig 1). EF1a was the most highly expressed HKG and RPS9 the least expressed HKG. GAPDH, tubulin and actin were moderately expressed when compared among other HKGs.


Validation of reference housekeeping genes for gene expression studies in western corn rootworm (Diabrotica virgifera virgifera).

Rodrigues TB, Barros Rodrigues T, Khajuria C, Wang H, Matz N, Cunha Cardoso D, Valicente FH, Zhou X, Siegfried B - PLoS ONE (2014)

Ct values of the five candidate reference genes in all four experiments.Black bars indicate an average between maximum (Max) and minimum (Min) Ct values.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214676&req=5

pone-0109825-g001: Ct values of the five candidate reference genes in all four experiments.Black bars indicate an average between maximum (Max) and minimum (Min) Ct values.
Mentions: The expression profiles of all qRT-PCR products for all primers and all experiments are shown in Figure 1. The expression levels of all HKGs were measured by the Ct value, which is the number of PCR cycles needed to reach a specific threshold level of detection and is inversely correlated with the quantity of initial RNA template. For qRT-PCR normalization, a moderately expressed reference gene is preferred because extremely high or low expression of a HKG could introduce variability to data analysis [31], so a standard Ct value range was analyzed for all four experiments (Fig 1). EF1a was the most highly expressed HKG and RPS9 the least expressed HKG. GAPDH, tubulin and actin were moderately expressed when compared among other HKGs.

Bottom Line: In general, normalization of results using a highly stable housekeeping gene (HKG) as an internal control is recommended and necessary.EF1a was also the most stable for Bt exposure and among different tissues.These results will enable researchers to use more accurate and reliable normalization of qRT-PCR data in WCR experiments.

View Article: PubMed Central - PubMed

Affiliation: CAPES Foundation, Ministry of Education of Brazil, Brasília, Federal District, Brazil, and Federal University of Lavras, Lavras, Minas Gerais, Brazil.

ABSTRACT
Quantitative Real-time PCR (qRT-PCR) is a powerful technique to investigate comparative gene expression. In general, normalization of results using a highly stable housekeeping gene (HKG) as an internal control is recommended and necessary. However, there are several reports suggesting that regulation of some HKGs is affected by different conditions. The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a serious pest of corn in the United States and Europe. The expression profile of target genes related to insecticide exposure, resistance, and RNA interference has become an important experimental technique for study of western corn rootworms; however, lack of information on reliable HKGs under different conditions makes the interpretation of qRT-PCR results difficult. In this study, four distinct algorithms (Genorm, NormFinder, BestKeeper and delta-CT) and five candidate HKGs to genes of reference (β-actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; β-tubulin; RPS9, ribosomal protein S9; EF1a, elongation factor-1α) were evaluated to determine the most reliable HKG under different experimental conditions including exposure to dsRNA and Bt toxins and among different tissues and developmental stages. Although all the HKGs tested exhibited relatively stable expression among the different treatments, some differences were noted. Among the five candidate reference genes evaluated, β-actin exhibited highly stable expression among different life stages. RPS9 exhibited the most similar pattern of expression among dsRNA treatments, and both experiments indicated that EF1a was the second most stable gene. EF1a was also the most stable for Bt exposure and among different tissues. These results will enable researchers to use more accurate and reliable normalization of qRT-PCR data in WCR experiments.

Show MeSH
Related in: MedlinePlus