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Evaluation and selection of candidate reference genes for normalization of quantitative RT-PCR in Withania somnifera (L.) Dunal.

Singh V, Kaul SC, Wadhwa R, Pati PK - PLoS ONE (2015)

Bottom Line: T-SAND was found to be a best reference gene for salt and salicylic acid (SA) treated samples, while 26S ribosomal RNA (26S), ubiquitin (UBQ) and beta-tubulin (TUB) were the most stably expressed genes under drought, biotic and cold treatment respectively.For abscisic acid (ABA) treated samples 18S-rRNA was found to stably expressed gene.The present work will significantly contribute to gene analysis studies in W. somnifera and facilitate in improving the quality of gene expression data in this plant as well as and other related plant species.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Guru Nanak Dev University, Amritsar-143005, Punjab, India.

ABSTRACT
Quantitative real-time PCR (qRT-PCR) is now globally used for accurate analysis of transcripts levels in plants. For reliable quantification of transcripts, identification of the best reference genes is a prerequisite in qRT-PCR analysis. Recently, Withania somnifera has attracted lot of attention due to its immense therapeutic potential. At present, biotechnological intervention for the improvement of this plant is being seriously pursued. In this background, it is important to have comprehensive studies on finding suitable reference genes for this high valued medicinal plant. In the present study, 11 candidate genes were evaluated for their expression stability under biotic (fungal disease), abiotic (wounding, salt, drought, heat and cold) stresses, in different plant tissues and in response to various plant growth regulators (methyl jasmonate, salicylic acid, abscisic acid). The data as analyzed by various software packages (geNorm, NormFinder, Bestkeeper and ΔCt method) suggested that cyclophilin (CYP) is a most stable gene under wounding, heat, methyl jasmonate, different tissues and all stress conditions. T-SAND was found to be a best reference gene for salt and salicylic acid (SA) treated samples, while 26S ribosomal RNA (26S), ubiquitin (UBQ) and beta-tubulin (TUB) were the most stably expressed genes under drought, biotic and cold treatment respectively. For abscisic acid (ABA) treated samples 18S-rRNA was found to stably expressed gene. Finally, the relative expression level of the three genes involved in the withanolide biosynthetic pathway was detected to validate the selection of reliable reference genes. The present work will significantly contribute to gene analysis studies in W. somnifera and facilitate in improving the quality of gene expression data in this plant as well as and other related plant species.

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Related in: MedlinePlus

Pairwise variation calculated by geNorm to determine the minimum number of reference genes required for accurate normalization in each experimental set.The cut off value is 0.15, below which the inclusion of an additional reference gene is not required.
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pone.0118860.g004: Pairwise variation calculated by geNorm to determine the minimum number of reference genes required for accurate normalization in each experimental set.The cut off value is 0.15, below which the inclusion of an additional reference gene is not required.

Mentions: To find out the optimal number of reference genes in each experimental condition, geNorm also calculate pairwise variation (V). Vandesompele and coworkers [27] usually recommends 0.15 as a cutoff value to determine the optimal number of reference genes. The inclusion of additional gene if V value is below this cutoff value is not considered. However, 0.15 is not an absolute cutoff value but rather an ideal value depending on the amount of genes and type of samples tested [32, 33]. Pairwise variation analysis showed that the optimal number of reference genes may be different for distinct stress samples. Analysis of the pairwise variation in cold, drought and biotic treatments revealed that the Vn+1 value is less than cutoff value of 0.15, indicating that only two reference genes would be sufficient for normalizing gene expression in these stress treatment (Fig. 4). For wounding treatment pairwise variation at the V2/3 value was 0.194, which is above the threshold of 0.15, and with the addition of next gene it drops down to stable value of 0.131, indicating that the proper normalization require at least three reference genes. Therefore, CYP, RPL2, and 26S can be used as reference genes to normalize gene expression data in wounding treated samples. With heat treated samples all genes pairwise variation or V number always remained above cutoff value of 0.15. On the other hand, four genes for SA and five genes for the MJ treated samples are required for the normalization. Among different tissue subset addition of seventh gene lower the V value below cutoff. Similarly addition of TUB (eighth gene) lower the V value in case of ABA treated samples. For all the samples taken together for analysis, it was observed that there was not a significant difference in the V numbers, but the instability was increased with the addition of TUA (V9/10) and 18S-rRNA (V10/11).


Evaluation and selection of candidate reference genes for normalization of quantitative RT-PCR in Withania somnifera (L.) Dunal.

Singh V, Kaul SC, Wadhwa R, Pati PK - PLoS ONE (2015)

Pairwise variation calculated by geNorm to determine the minimum number of reference genes required for accurate normalization in each experimental set.The cut off value is 0.15, below which the inclusion of an additional reference gene is not required.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359125&req=5

pone.0118860.g004: Pairwise variation calculated by geNorm to determine the minimum number of reference genes required for accurate normalization in each experimental set.The cut off value is 0.15, below which the inclusion of an additional reference gene is not required.
Mentions: To find out the optimal number of reference genes in each experimental condition, geNorm also calculate pairwise variation (V). Vandesompele and coworkers [27] usually recommends 0.15 as a cutoff value to determine the optimal number of reference genes. The inclusion of additional gene if V value is below this cutoff value is not considered. However, 0.15 is not an absolute cutoff value but rather an ideal value depending on the amount of genes and type of samples tested [32, 33]. Pairwise variation analysis showed that the optimal number of reference genes may be different for distinct stress samples. Analysis of the pairwise variation in cold, drought and biotic treatments revealed that the Vn+1 value is less than cutoff value of 0.15, indicating that only two reference genes would be sufficient for normalizing gene expression in these stress treatment (Fig. 4). For wounding treatment pairwise variation at the V2/3 value was 0.194, which is above the threshold of 0.15, and with the addition of next gene it drops down to stable value of 0.131, indicating that the proper normalization require at least three reference genes. Therefore, CYP, RPL2, and 26S can be used as reference genes to normalize gene expression data in wounding treated samples. With heat treated samples all genes pairwise variation or V number always remained above cutoff value of 0.15. On the other hand, four genes for SA and five genes for the MJ treated samples are required for the normalization. Among different tissue subset addition of seventh gene lower the V value below cutoff. Similarly addition of TUB (eighth gene) lower the V value in case of ABA treated samples. For all the samples taken together for analysis, it was observed that there was not a significant difference in the V numbers, but the instability was increased with the addition of TUA (V9/10) and 18S-rRNA (V10/11).

Bottom Line: T-SAND was found to be a best reference gene for salt and salicylic acid (SA) treated samples, while 26S ribosomal RNA (26S), ubiquitin (UBQ) and beta-tubulin (TUB) were the most stably expressed genes under drought, biotic and cold treatment respectively.For abscisic acid (ABA) treated samples 18S-rRNA was found to stably expressed gene.The present work will significantly contribute to gene analysis studies in W. somnifera and facilitate in improving the quality of gene expression data in this plant as well as and other related plant species.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Guru Nanak Dev University, Amritsar-143005, Punjab, India.

ABSTRACT
Quantitative real-time PCR (qRT-PCR) is now globally used for accurate analysis of transcripts levels in plants. For reliable quantification of transcripts, identification of the best reference genes is a prerequisite in qRT-PCR analysis. Recently, Withania somnifera has attracted lot of attention due to its immense therapeutic potential. At present, biotechnological intervention for the improvement of this plant is being seriously pursued. In this background, it is important to have comprehensive studies on finding suitable reference genes for this high valued medicinal plant. In the present study, 11 candidate genes were evaluated for their expression stability under biotic (fungal disease), abiotic (wounding, salt, drought, heat and cold) stresses, in different plant tissues and in response to various plant growth regulators (methyl jasmonate, salicylic acid, abscisic acid). The data as analyzed by various software packages (geNorm, NormFinder, Bestkeeper and ΔCt method) suggested that cyclophilin (CYP) is a most stable gene under wounding, heat, methyl jasmonate, different tissues and all stress conditions. T-SAND was found to be a best reference gene for salt and salicylic acid (SA) treated samples, while 26S ribosomal RNA (26S), ubiquitin (UBQ) and beta-tubulin (TUB) were the most stably expressed genes under drought, biotic and cold treatment respectively. For abscisic acid (ABA) treated samples 18S-rRNA was found to stably expressed gene. Finally, the relative expression level of the three genes involved in the withanolide biosynthetic pathway was detected to validate the selection of reliable reference genes. The present work will significantly contribute to gene analysis studies in W. somnifera and facilitate in improving the quality of gene expression data in this plant as well as and other related plant species.

Show MeSH
Related in: MedlinePlus