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Identification of reference genes for quantitative expression analysis of microRNAs and mRNAs in barley under various stress conditions.

Ferdous J, Li Y, Reid N, Langridge P, Shi BJ, Tricker PJ - PLoS ONE (2015)

Bottom Line: We found that ADP, snoR14 and snoR23 were ranked as the best of these candidates across diverse samples.Additionally, we found that miR168 was a suitable reference gene for expression analysis in barley.Finally, we validated the performance of our stable and unstable candidate reference genes for both mRNA and miRNA qPCR data normalization under different stress conditions and demonstrated the superiority of the stable candidates.

View Article: PubMed Central - PubMed

Affiliation: Australian Centre for Plant Functional Genomics, University of Adelaide, Hartley Grove, Urrbrae, South Australia 5064, Australia.

ABSTRACT
For accurate and reliable gene expression analysis using quantitative real-time reverse transcription PCR (qPCR), the selection of appropriate reference genes as an internal control for normalization is crucial. We hypothesized that non-coding, small nucleolar RNAs (snoRNAs)would be stably expressed in different barley varieties and under different experimental treatments,in different tissues and at different developmental stages of plant growth and therefore might prove to be suitable reference genes for expression analysis of both microRNAs (miRNAs)and mRNAs. In this study, we examined the expression stability of ten candidate reference genes in six barley genotypes under five experimental stresses, drought, fungal infection,boron toxicity, nutrient deficiency and salinity. We compared four commonly used housekeeping genes; Actin (ACT), alpha-Tubulin (α-TUB), Glycolytic glyceraldehyde-3-phosphate dehydrogenase(GAPDH), ADP-ribosylation factor 1-like protein (ADP), four snoRNAs; (U18,U61, snoR14 and snoR23) and two microRNAs (miR168, miR159) as candidate reference genes. We found that ADP, snoR14 and snoR23 were ranked as the best of these candidates across diverse samples. Additionally, we found that miR168 was a suitable reference gene for expression analysis in barley. Finally, we validated the performance of our stable and unstable candidate reference genes for both mRNA and miRNA qPCR data normalization under different stress conditions and demonstrated the superiority of the stable candidates. Our data demonstrate the suitability of barley snoRNAs and miRNAs as potential reference genes form iRNA and mRNA qPCR data normalization under different stress treatments [corrected].

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Validation of putatively stable reference genes.Comparison of relative expression of (A, B, C, D, E) SUPEROXIDE DISMUTASE (AK363344.1) and (F, G, H, I, J) miR5048 (MIMAT0020544) in drought-treated, boron treated, fungal infected, nitrate treated and salt treated samples and their respective controls by qPCR when normalized to group1 (a combined group of three stable reference genes; snoR14, ADP, snoR23) and group 2 (a combined group commonly used housekeeping genes; ACT, α-TUB, GAPDH). The error bars indicate the standard deviation of the mean. Statistical analysis by t-test. (Inset in 5F) The relative expression of miR5048 from deep sequencing of drought-stressed and well-watered barley leaves. Reads per million (RPM) of 1 was the highest number of counts.
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pone.0118503.g005: Validation of putatively stable reference genes.Comparison of relative expression of (A, B, C, D, E) SUPEROXIDE DISMUTASE (AK363344.1) and (F, G, H, I, J) miR5048 (MIMAT0020544) in drought-treated, boron treated, fungal infected, nitrate treated and salt treated samples and their respective controls by qPCR when normalized to group1 (a combined group of three stable reference genes; snoR14, ADP, snoR23) and group 2 (a combined group commonly used housekeeping genes; ACT, α-TUB, GAPDH). The error bars indicate the standard deviation of the mean. Statistical analysis by t-test. (Inset in 5F) The relative expression of miR5048 from deep sequencing of drought-stressed and well-watered barley leaves. Reads per million (RPM) of 1 was the highest number of counts.

Mentions: To validate the stability of reference genes, we used two groups for normalization. Group 1 denotes the three putatively stable reference genes, ADP, snoR14 and snoR23, suggested by the consensus ranking, and group 2 denotes the three commonly used HKGs, ACT, α-TUB and GAPDH. For each experimental condition, the expression of SUPEROXIDE DISMUTASE was normalized to group 1 and group 2 in the treated and control samples (Fig. 5). The formation of reactive oxygen species (ROS) in plants is triggered by various environmental stresses, such as drought, nutrient deficiency, nutrient toxicity, salinity and pathogen attack [46, 47]. SUPEROXIDE DISMUTASE is one of the first line of defence antioxidant enzymes. Though its expression is not broadly reported for each particular stress condition, it is well-known to be up-regulated under drought stress in plant species including barley [48–55]. As expected, normalized expression of SUPEROXIDE DISMUTASE to group 1 and group 2 resulted in lower expression under well-watered (control) than under drought-treated samples (Fig. 5A). However, the expression difference between the treated and control samples was greater when normalized with group 1 candidates (Fig. 5A). In the boron treatment experiment, both normalizer groups showed similar results where SUPEROXIDE DISMUTASE was significantly up-regulated in the treated samples (Fig. 5B). In the fungal infected samples the expression level of SUPEROXIDE DISMUTASE was reduced compared to the control samples when normalized to group 1. However, when normalized to group 2 the expression level of SUPEROXIDE DISMUTASE was not significantly different (Fig. 5C). In the nitrate and salt treatment samples, group 1 normalization resulted in significantly higher SUPEROXIDE DISMUTASE expression than in the control samples (Fig. 5D & E). However, in both of these experimental conditions, group 2 normalization did not show any significant difference of SUPEROXIDE DISMUTASE expression between the treated and control samples (Fig. 5D & E).


Identification of reference genes for quantitative expression analysis of microRNAs and mRNAs in barley under various stress conditions.

Ferdous J, Li Y, Reid N, Langridge P, Shi BJ, Tricker PJ - PLoS ONE (2015)

Validation of putatively stable reference genes.Comparison of relative expression of (A, B, C, D, E) SUPEROXIDE DISMUTASE (AK363344.1) and (F, G, H, I, J) miR5048 (MIMAT0020544) in drought-treated, boron treated, fungal infected, nitrate treated and salt treated samples and their respective controls by qPCR when normalized to group1 (a combined group of three stable reference genes; snoR14, ADP, snoR23) and group 2 (a combined group commonly used housekeeping genes; ACT, α-TUB, GAPDH). The error bars indicate the standard deviation of the mean. Statistical analysis by t-test. (Inset in 5F) The relative expression of miR5048 from deep sequencing of drought-stressed and well-watered barley leaves. Reads per million (RPM) of 1 was the highest number of counts.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4368757&req=5

pone.0118503.g005: Validation of putatively stable reference genes.Comparison of relative expression of (A, B, C, D, E) SUPEROXIDE DISMUTASE (AK363344.1) and (F, G, H, I, J) miR5048 (MIMAT0020544) in drought-treated, boron treated, fungal infected, nitrate treated and salt treated samples and their respective controls by qPCR when normalized to group1 (a combined group of three stable reference genes; snoR14, ADP, snoR23) and group 2 (a combined group commonly used housekeeping genes; ACT, α-TUB, GAPDH). The error bars indicate the standard deviation of the mean. Statistical analysis by t-test. (Inset in 5F) The relative expression of miR5048 from deep sequencing of drought-stressed and well-watered barley leaves. Reads per million (RPM) of 1 was the highest number of counts.
Mentions: To validate the stability of reference genes, we used two groups for normalization. Group 1 denotes the three putatively stable reference genes, ADP, snoR14 and snoR23, suggested by the consensus ranking, and group 2 denotes the three commonly used HKGs, ACT, α-TUB and GAPDH. For each experimental condition, the expression of SUPEROXIDE DISMUTASE was normalized to group 1 and group 2 in the treated and control samples (Fig. 5). The formation of reactive oxygen species (ROS) in plants is triggered by various environmental stresses, such as drought, nutrient deficiency, nutrient toxicity, salinity and pathogen attack [46, 47]. SUPEROXIDE DISMUTASE is one of the first line of defence antioxidant enzymes. Though its expression is not broadly reported for each particular stress condition, it is well-known to be up-regulated under drought stress in plant species including barley [48–55]. As expected, normalized expression of SUPEROXIDE DISMUTASE to group 1 and group 2 resulted in lower expression under well-watered (control) than under drought-treated samples (Fig. 5A). However, the expression difference between the treated and control samples was greater when normalized with group 1 candidates (Fig. 5A). In the boron treatment experiment, both normalizer groups showed similar results where SUPEROXIDE DISMUTASE was significantly up-regulated in the treated samples (Fig. 5B). In the fungal infected samples the expression level of SUPEROXIDE DISMUTASE was reduced compared to the control samples when normalized to group 1. However, when normalized to group 2 the expression level of SUPEROXIDE DISMUTASE was not significantly different (Fig. 5C). In the nitrate and salt treatment samples, group 1 normalization resulted in significantly higher SUPEROXIDE DISMUTASE expression than in the control samples (Fig. 5D & E). However, in both of these experimental conditions, group 2 normalization did not show any significant difference of SUPEROXIDE DISMUTASE expression between the treated and control samples (Fig. 5D & E).

Bottom Line: We found that ADP, snoR14 and snoR23 were ranked as the best of these candidates across diverse samples.Additionally, we found that miR168 was a suitable reference gene for expression analysis in barley.Finally, we validated the performance of our stable and unstable candidate reference genes for both mRNA and miRNA qPCR data normalization under different stress conditions and demonstrated the superiority of the stable candidates.

View Article: PubMed Central - PubMed

Affiliation: Australian Centre for Plant Functional Genomics, University of Adelaide, Hartley Grove, Urrbrae, South Australia 5064, Australia.

ABSTRACT
For accurate and reliable gene expression analysis using quantitative real-time reverse transcription PCR (qPCR), the selection of appropriate reference genes as an internal control for normalization is crucial. We hypothesized that non-coding, small nucleolar RNAs (snoRNAs)would be stably expressed in different barley varieties and under different experimental treatments,in different tissues and at different developmental stages of plant growth and therefore might prove to be suitable reference genes for expression analysis of both microRNAs (miRNAs)and mRNAs. In this study, we examined the expression stability of ten candidate reference genes in six barley genotypes under five experimental stresses, drought, fungal infection,boron toxicity, nutrient deficiency and salinity. We compared four commonly used housekeeping genes; Actin (ACT), alpha-Tubulin (α-TUB), Glycolytic glyceraldehyde-3-phosphate dehydrogenase(GAPDH), ADP-ribosylation factor 1-like protein (ADP), four snoRNAs; (U18,U61, snoR14 and snoR23) and two microRNAs (miR168, miR159) as candidate reference genes. We found that ADP, snoR14 and snoR23 were ranked as the best of these candidates across diverse samples. Additionally, we found that miR168 was a suitable reference gene for expression analysis in barley. Finally, we validated the performance of our stable and unstable candidate reference genes for both mRNA and miRNA qPCR data normalization under different stress conditions and demonstrated the superiority of the stable candidates. Our data demonstrate the suitability of barley snoRNAs and miRNAs as potential reference genes form iRNA and mRNA qPCR data normalization under different stress treatments [corrected].

Show MeSH
Related in: MedlinePlus