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Validation of novel reference genes for reverse transcription quantitative real-time PCR in drought-stressed sugarcane.

Silva RL, Silva MD, Ferreira Neto JR, de Nardi CH, Chabregas SM, Burnquist WL, Kahl G, Benko-Iseppon AM, Kido EA - ScientificWorldJournal (2014)

Bottom Line: Their suitability as reference genes validated the expression profiles of two targets (AS and PFP α1), related to SuperSAGE unitags, in agreement with results revealed by previous in silico analysis.The other two sugarcane unitags (ACC oxidase and PIP1-1), after salt stress (100 mM NaCl), presented their expressions validated in the same way.In conclusion, these reference genes will be useful for dissecting gene expression in sugarcane roots under abiotic stress, especially in transcriptomic studies using SuperSAGE or RNAseq approaches.

View Article: PubMed Central - PubMed

Affiliation: Federal University of Pernambuco (UFPE/CCB/Genética), 50670-420 Recife, PE, Brazil.

ABSTRACT
One of the most challenging aspects of RT-qPCR data analysis is the identification of reliable reference genes. Ideally, they should be neither induced nor repressed under different experimental conditions. To date, few reference genes have been adequately studied for sugarcane (Saccharum spp.) using statistical approaches. In this work, six candidate genes ( αTUB, GAPDH, H1, SAMDC, UBQ, and 25S rRNA) were tested for gene expression normalization of sugarcane root tissues from drought-tolerant and -sensitive accessions after continuous dehydration (24 h). By undergoing different approaches (GeNorm, NormFinder, and BestKeeper), it was shown that most of them could be used in combinations for normalization purposes, with the exception of SAMDC. Nevertheless three of them (H1, αTUB, and GAPDH) were considered the most reliable reference genes. Their suitability as reference genes validated the expression profiles of two targets (AS and PFP α1), related to SuperSAGE unitags, in agreement with results revealed by previous in silico analysis. The other two sugarcane unitags (ACC oxidase and PIP1-1), after salt stress (100 mM NaCl), presented their expressions validated in the same way. In conclusion, these reference genes will be useful for dissecting gene expression in sugarcane roots under abiotic stress, especially in transcriptomic studies using SuperSAGE or RNAseq approaches.

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Related in: MedlinePlus

Average gene expression stability values (M) of six sugarcane potential reference genes (αTUB: alpha-tubulin; GAPDH: glyceraldehyde 3 phosphate dehydrogenase; H1: histone H1; SAMDC: S-adenosylmethionine decarboxylase; UBQ: ubiquitin; 25S rRNA: 25S ribosomal RNA) based on the GeNorm analysis [17].
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Related In: Results  -  Collection


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fig1: Average gene expression stability values (M) of six sugarcane potential reference genes (αTUB: alpha-tubulin; GAPDH: glyceraldehyde 3 phosphate dehydrogenase; H1: histone H1; SAMDC: S-adenosylmethionine decarboxylase; UBQ: ubiquitin; 25S rRNA: 25S ribosomal RNA) based on the GeNorm analysis [17].

Mentions: Considering the average expression stability values (M-value), αTUB (M = 0.61), GAPDH (M = 0.62), and histone H1 (M = 0.63) were the most stable genes while SAMDC represented the most variable (M = 0.87) gene. However, all of them showed an expressive high stability with M-values below 1 (Figure 1), suggesting that all the six candidates may be adequate for normalizing gene expression data under the conditions used in the present work. Besides, based on the pairwise variation (V) data (Figure 2), it was possible to determine the optimal number of reference genes required for the relative quantification analysis and to investigate the effect of gene addition in this normalization. The data suggested that the addition to the two most stable genes (αTUB and GAPDH) considering a third gene (V2/3 = 0.15; Figure 2), a fourth (V3/4 = 0.14), or even more genes (V4/5 and V5/6; Figure 2) still exhibited desired values (below 0.15 as proposed by Vandesompele et al. [17]). To normalize the gene expression in the above mentioned sugarcane samples, αTUB, GAPDH, and H1 seem to be sufficient (Figure 1).


Validation of novel reference genes for reverse transcription quantitative real-time PCR in drought-stressed sugarcane.

Silva RL, Silva MD, Ferreira Neto JR, de Nardi CH, Chabregas SM, Burnquist WL, Kahl G, Benko-Iseppon AM, Kido EA - ScientificWorldJournal (2014)

Average gene expression stability values (M) of six sugarcane potential reference genes (αTUB: alpha-tubulin; GAPDH: glyceraldehyde 3 phosphate dehydrogenase; H1: histone H1; SAMDC: S-adenosylmethionine decarboxylase; UBQ: ubiquitin; 25S rRNA: 25S ribosomal RNA) based on the GeNorm analysis [17].
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4060590&req=5

fig1: Average gene expression stability values (M) of six sugarcane potential reference genes (αTUB: alpha-tubulin; GAPDH: glyceraldehyde 3 phosphate dehydrogenase; H1: histone H1; SAMDC: S-adenosylmethionine decarboxylase; UBQ: ubiquitin; 25S rRNA: 25S ribosomal RNA) based on the GeNorm analysis [17].
Mentions: Considering the average expression stability values (M-value), αTUB (M = 0.61), GAPDH (M = 0.62), and histone H1 (M = 0.63) were the most stable genes while SAMDC represented the most variable (M = 0.87) gene. However, all of them showed an expressive high stability with M-values below 1 (Figure 1), suggesting that all the six candidates may be adequate for normalizing gene expression data under the conditions used in the present work. Besides, based on the pairwise variation (V) data (Figure 2), it was possible to determine the optimal number of reference genes required for the relative quantification analysis and to investigate the effect of gene addition in this normalization. The data suggested that the addition to the two most stable genes (αTUB and GAPDH) considering a third gene (V2/3 = 0.15; Figure 2), a fourth (V3/4 = 0.14), or even more genes (V4/5 and V5/6; Figure 2) still exhibited desired values (below 0.15 as proposed by Vandesompele et al. [17]). To normalize the gene expression in the above mentioned sugarcane samples, αTUB, GAPDH, and H1 seem to be sufficient (Figure 1).

Bottom Line: Their suitability as reference genes validated the expression profiles of two targets (AS and PFP α1), related to SuperSAGE unitags, in agreement with results revealed by previous in silico analysis.The other two sugarcane unitags (ACC oxidase and PIP1-1), after salt stress (100 mM NaCl), presented their expressions validated in the same way.In conclusion, these reference genes will be useful for dissecting gene expression in sugarcane roots under abiotic stress, especially in transcriptomic studies using SuperSAGE or RNAseq approaches.

View Article: PubMed Central - PubMed

Affiliation: Federal University of Pernambuco (UFPE/CCB/Genética), 50670-420 Recife, PE, Brazil.

ABSTRACT
One of the most challenging aspects of RT-qPCR data analysis is the identification of reliable reference genes. Ideally, they should be neither induced nor repressed under different experimental conditions. To date, few reference genes have been adequately studied for sugarcane (Saccharum spp.) using statistical approaches. In this work, six candidate genes ( αTUB, GAPDH, H1, SAMDC, UBQ, and 25S rRNA) were tested for gene expression normalization of sugarcane root tissues from drought-tolerant and -sensitive accessions after continuous dehydration (24 h). By undergoing different approaches (GeNorm, NormFinder, and BestKeeper), it was shown that most of them could be used in combinations for normalization purposes, with the exception of SAMDC. Nevertheless three of them (H1, αTUB, and GAPDH) were considered the most reliable reference genes. Their suitability as reference genes validated the expression profiles of two targets (AS and PFP α1), related to SuperSAGE unitags, in agreement with results revealed by previous in silico analysis. The other two sugarcane unitags (ACC oxidase and PIP1-1), after salt stress (100 mM NaCl), presented their expressions validated in the same way. In conclusion, these reference genes will be useful for dissecting gene expression in sugarcane roots under abiotic stress, especially in transcriptomic studies using SuperSAGE or RNAseq approaches.

Show MeSH
Related in: MedlinePlus