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Identification of candidate reference genes in perennial ryegrass for quantitative RT-PCR under various abiotic stress conditions.

Huang L, Yan H, Jiang X, Yin G, Zhang X, Qi X, Zhang Y, Yan Y, Ma X, Peng Y - PLoS ONE (2014)

Bottom Line: The results are consistent with the idea that the best reference genes depend on the stress treatment under investigation.Eukaryotic initiation factor 4 alpha (eIF4A), Transcription elongation factor 1 (TEF1) and Tat binding protein-1 (TBP-1) were the three most stably expressed genes under drought stress and were also the three best genes for studying salt stress. eIF4A, TBP-1, and Ubiquitin-conjugating enzyme (E2) were the most suitable reference genes to study heat stress, while eIF4A, TEF1, and E2 were the three best reference genes for studying the effects of ABA.The stability of expression in these reference genes will enable better normalization and quantification of the transcript levels for studies of gene expression in such studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Grassland Science, Animal Science and Technology College, Sichuan Agricultural University, Ya'an, Sichuan, China.

ABSTRACT

Background: Quantitative real-time reverse-transcriptase PCR (qRT-PCR) is an important technique for analyzing differences in gene expression due to its sensitivity, accuracy and specificity. However, the stability of the expression of reference genes is necessary to ensure accurate qRT-PCR assessment of expression in genes of interest. Perennial ryegrass (Lolium perenne L.) is important forage and turf grass species in temperate regions, but the expression stability of its reference genes under various stresses has not been well-studied.

Methodology/principal findings: In this study, 11 candidate reference genes were evaluated for use as controls in qRT-PCR to quantify gene expression in perennial ryegrass under drought, high salinity, heat, waterlogging, and ABA (abscisic acid) treatments. Four approaches--Delta CT, geNorm, BestKeeper and Normfinder were used to determine the stability of expression in these reference genes. The results are consistent with the idea that the best reference genes depend on the stress treatment under investigation. Eukaryotic initiation factor 4 alpha (eIF4A), Transcription elongation factor 1 (TEF1) and Tat binding protein-1 (TBP-1) were the three most stably expressed genes under drought stress and were also the three best genes for studying salt stress. eIF4A, TBP-1, and Ubiquitin-conjugating enzyme (E2) were the most suitable reference genes to study heat stress, while eIF4A, TEF1, and E2 were the three best reference genes for studying the effects of ABA. Finally, Ubiquitin (UBQ), TEF1, and eIF4A were the three best reference genes for waterlogging treatments.

Conclusions/significance: These results will be helpful in choosing the best reference genes for use in studies related to various abiotic stresses in perennial ryegrass. The stability of expression in these reference genes will enable better normalization and quantification of the transcript levels for studies of gene expression in such studies.

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Related in: MedlinePlus

Expression levels of SOD (a) and POD (b) in different time (Days 0, 3, 6, and 9) of perennial ryegrass leaves under drought stress.Genes were normalized to highly stable (eIF4A) and unstable (60S) reference genes. Error bars indicate one standard error of the mean.
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pone-0093724-g004: Expression levels of SOD (a) and POD (b) in different time (Days 0, 3, 6, and 9) of perennial ryegrass leaves under drought stress.Genes were normalized to highly stable (eIF4A) and unstable (60S) reference genes. Error bars indicate one standard error of the mean.

Mentions: To validate the performance of the reference genes identified in this study on known abiotic-stress inducible genes, we quantified the expression of SOD and POD genes which are up-regulated under dehydration drought stress [39]–[41]. After quantifying, a representative least stable reference gene (60S) and a representative most stable reference gene (eIF4A) were used to normalize their expression. As shown in Figure 4, using 60S for normalization, suggests that both SOD and POD genes are induced ten or six fold, respectively, on both Day 3 and Day 6 after initiation of drought treatment. In contrast, using eIF4A as the reference gene reveals greater overall fold changes in expression of SOD and POD compared to Day 0 and greater expression of both genes on Day 6 than on Day 3. Furthermore, if 60S was used as reference gene, we would have consistently failed to detect drought-induced gene expression changes in perennial ryegrass leaves for SOD (Day 3 to Day 6) and POD (between Days 3, 6, and 9) that were clearly identified when normalization was carried out with respect to eIF4A (Figure 4). Gel electrophoresis of PCR products also showed (Figure 5) that eIF4A was expressed more stable than 60S under drought treatment.


Identification of candidate reference genes in perennial ryegrass for quantitative RT-PCR under various abiotic stress conditions.

Huang L, Yan H, Jiang X, Yin G, Zhang X, Qi X, Zhang Y, Yan Y, Ma X, Peng Y - PLoS ONE (2014)

Expression levels of SOD (a) and POD (b) in different time (Days 0, 3, 6, and 9) of perennial ryegrass leaves under drought stress.Genes were normalized to highly stable (eIF4A) and unstable (60S) reference genes. Error bars indicate one standard error of the mean.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3974806&req=5

pone-0093724-g004: Expression levels of SOD (a) and POD (b) in different time (Days 0, 3, 6, and 9) of perennial ryegrass leaves under drought stress.Genes were normalized to highly stable (eIF4A) and unstable (60S) reference genes. Error bars indicate one standard error of the mean.
Mentions: To validate the performance of the reference genes identified in this study on known abiotic-stress inducible genes, we quantified the expression of SOD and POD genes which are up-regulated under dehydration drought stress [39]–[41]. After quantifying, a representative least stable reference gene (60S) and a representative most stable reference gene (eIF4A) were used to normalize their expression. As shown in Figure 4, using 60S for normalization, suggests that both SOD and POD genes are induced ten or six fold, respectively, on both Day 3 and Day 6 after initiation of drought treatment. In contrast, using eIF4A as the reference gene reveals greater overall fold changes in expression of SOD and POD compared to Day 0 and greater expression of both genes on Day 6 than on Day 3. Furthermore, if 60S was used as reference gene, we would have consistently failed to detect drought-induced gene expression changes in perennial ryegrass leaves for SOD (Day 3 to Day 6) and POD (between Days 3, 6, and 9) that were clearly identified when normalization was carried out with respect to eIF4A (Figure 4). Gel electrophoresis of PCR products also showed (Figure 5) that eIF4A was expressed more stable than 60S under drought treatment.

Bottom Line: The results are consistent with the idea that the best reference genes depend on the stress treatment under investigation.Eukaryotic initiation factor 4 alpha (eIF4A), Transcription elongation factor 1 (TEF1) and Tat binding protein-1 (TBP-1) were the three most stably expressed genes under drought stress and were also the three best genes for studying salt stress. eIF4A, TBP-1, and Ubiquitin-conjugating enzyme (E2) were the most suitable reference genes to study heat stress, while eIF4A, TEF1, and E2 were the three best reference genes for studying the effects of ABA.The stability of expression in these reference genes will enable better normalization and quantification of the transcript levels for studies of gene expression in such studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Grassland Science, Animal Science and Technology College, Sichuan Agricultural University, Ya'an, Sichuan, China.

ABSTRACT

Background: Quantitative real-time reverse-transcriptase PCR (qRT-PCR) is an important technique for analyzing differences in gene expression due to its sensitivity, accuracy and specificity. However, the stability of the expression of reference genes is necessary to ensure accurate qRT-PCR assessment of expression in genes of interest. Perennial ryegrass (Lolium perenne L.) is important forage and turf grass species in temperate regions, but the expression stability of its reference genes under various stresses has not been well-studied.

Methodology/principal findings: In this study, 11 candidate reference genes were evaluated for use as controls in qRT-PCR to quantify gene expression in perennial ryegrass under drought, high salinity, heat, waterlogging, and ABA (abscisic acid) treatments. Four approaches--Delta CT, geNorm, BestKeeper and Normfinder were used to determine the stability of expression in these reference genes. The results are consistent with the idea that the best reference genes depend on the stress treatment under investigation. Eukaryotic initiation factor 4 alpha (eIF4A), Transcription elongation factor 1 (TEF1) and Tat binding protein-1 (TBP-1) were the three most stably expressed genes under drought stress and were also the three best genes for studying salt stress. eIF4A, TBP-1, and Ubiquitin-conjugating enzyme (E2) were the most suitable reference genes to study heat stress, while eIF4A, TEF1, and E2 were the three best reference genes for studying the effects of ABA. Finally, Ubiquitin (UBQ), TEF1, and eIF4A were the three best reference genes for waterlogging treatments.

Conclusions/significance: These results will be helpful in choosing the best reference genes for use in studies related to various abiotic stresses in perennial ryegrass. The stability of expression in these reference genes will enable better normalization and quantification of the transcript levels for studies of gene expression in such studies.

Show MeSH
Related in: MedlinePlus