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Selection and validation of reference genes for qRT-PCR expression analysis of candidate genes involved in olfactory communication in the butterfly Bicyclus anynana.

Arun A, Baumlé V, Amelot G, Nieberding CM - PLoS ONE (2015)

Bottom Line: The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm.Both methods identified RpS8 as the best suitable reference gene for expression data normalization.We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana.

View Article: PubMed Central - PubMed

Affiliation: Evolutionary Ecology and Genetics group, Biodiversity Research Centre, Earth and Life Institute, Université catholique de Louvain, Croix du Sud 4, Louvain-la-Neuve, Belgium.

ABSTRACT
Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae), two developmental stages (pupal and adult) and two sexes (male and female), all of which were subjected to two food treatments (food stress and control feeding ad libitum). The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the expression profile of the target candidate genes.

No MeSH data available.


Measures of expression stability (M value).geNorm analysis and ranking of 12 candidate reference genes in (A) all tissues (B) control tissues and (C) food stressed tissues. Suitable reference genes are assigned M values below 0.5 (dotted line).
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pone.0120401.g002: Measures of expression stability (M value).geNorm analysis and ranking of 12 candidate reference genes in (A) all tissues (B) control tissues and (C) food stressed tissues. Suitable reference genes are assigned M values below 0.5 (dotted line).

Mentions: Second, we used geNorm, for which Vandesompele et al [35] strongly recommends a threshold expression stability M value of 0.5. When M values from both control and food stressed treatments were collectively evaluated, four out of twelve reference genes showed M value lower than 0.5, and RpS8 had the lowest M value of 0.35 and therefore was the best reference gene (Fig. 2A). In the control treatment, 6 out of 12 reference genes had a M value lower than 0.5 (Fig. 2B) while in the food stressed treatment, five out of twelve tested reference genes had a M value lower than 0.5 (Fig. 2C), among which, RpS8 was each time the most stable reference gene with an acceptable M value of 0.31. Our earlier conclusion that ACT3 was the least stable reference gene was also supported bygeNorm analysis.


Selection and validation of reference genes for qRT-PCR expression analysis of candidate genes involved in olfactory communication in the butterfly Bicyclus anynana.

Arun A, Baumlé V, Amelot G, Nieberding CM - PLoS ONE (2015)

Measures of expression stability (M value).geNorm analysis and ranking of 12 candidate reference genes in (A) all tissues (B) control tissues and (C) food stressed tissues. Suitable reference genes are assigned M values below 0.5 (dotted line).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4368739&req=5

pone.0120401.g002: Measures of expression stability (M value).geNorm analysis and ranking of 12 candidate reference genes in (A) all tissues (B) control tissues and (C) food stressed tissues. Suitable reference genes are assigned M values below 0.5 (dotted line).
Mentions: Second, we used geNorm, for which Vandesompele et al [35] strongly recommends a threshold expression stability M value of 0.5. When M values from both control and food stressed treatments were collectively evaluated, four out of twelve reference genes showed M value lower than 0.5, and RpS8 had the lowest M value of 0.35 and therefore was the best reference gene (Fig. 2A). In the control treatment, 6 out of 12 reference genes had a M value lower than 0.5 (Fig. 2B) while in the food stressed treatment, five out of twelve tested reference genes had a M value lower than 0.5 (Fig. 2C), among which, RpS8 was each time the most stable reference gene with an acceptable M value of 0.31. Our earlier conclusion that ACT3 was the least stable reference gene was also supported bygeNorm analysis.

Bottom Line: The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm.Both methods identified RpS8 as the best suitable reference gene for expression data normalization.We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana.

View Article: PubMed Central - PubMed

Affiliation: Evolutionary Ecology and Genetics group, Biodiversity Research Centre, Earth and Life Institute, Université catholique de Louvain, Croix du Sud 4, Louvain-la-Neuve, Belgium.

ABSTRACT
Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae), two developmental stages (pupal and adult) and two sexes (male and female), all of which were subjected to two food treatments (food stress and control feeding ad libitum). The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the expression profile of the target candidate genes.

No MeSH data available.