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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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Relative expression level of withanolides biosynthesis genes in response to SA treatment.Transcript level relative quantification of HMGR (A), CAS (B) and P450 (C). The most stable genes recommended for SA treatment (T-SAND and UBQ) and the least stable gene GAPDH were used for normalization. For T-SAND and UBQ geometric mean was calculated and used for normalization of expression. Error bars show standard deviation calculated from three biological replicates.
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pone.0118860.g005: Relative expression level of withanolides biosynthesis genes in response to SA treatment.Transcript level relative quantification of HMGR (A), CAS (B) and P450 (C). The most stable genes recommended for SA treatment (T-SAND and UBQ) and the least stable gene GAPDH were used for normalization. For T-SAND and UBQ geometric mean was calculated and used for normalization of expression. Error bars show standard deviation calculated from three biological replicates.

Mentions: In order to further evaluate the expression stability of selected reference genes, we analyzed the expression of three genes that play key role in withanolide biosynthesis pathway. The expression of HMGR and CAS was not significantly affected by treatment with SA. The results reveal that the expression level of HMGR was highest after 12 h of treatment and then decline thereafter. The relative expression of CAS showed a variable pattern of expression where it showed significant upregulation after 12 and 48 h of treatment. Expression analysis of P450 showed upregulation after SA treatment and reached maximum after 48 h (Fig. 5).


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)

Relative expression level of withanolides biosynthesis genes in response to SA treatment.Transcript level relative quantification of HMGR (A), CAS (B) and P450 (C). The most stable genes recommended for SA treatment (T-SAND and UBQ) and the least stable gene GAPDH were used for normalization. For T-SAND and UBQ geometric mean was calculated and used for normalization of expression. Error bars show standard deviation calculated from three biological replicates.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118860.g005: Relative expression level of withanolides biosynthesis genes in response to SA treatment.Transcript level relative quantification of HMGR (A), CAS (B) and P450 (C). The most stable genes recommended for SA treatment (T-SAND and UBQ) and the least stable gene GAPDH were used for normalization. For T-SAND and UBQ geometric mean was calculated and used for normalization of expression. Error bars show standard deviation calculated from three biological replicates.
Mentions: In order to further evaluate the expression stability of selected reference genes, we analyzed the expression of three genes that play key role in withanolide biosynthesis pathway. The expression of HMGR and CAS was not significantly affected by treatment with SA. The results reveal that the expression level of HMGR was highest after 12 h of treatment and then decline thereafter. The relative expression of CAS showed a variable pattern of expression where it showed significant upregulation after 12 and 48 h of treatment. Expression analysis of P450 showed upregulation after SA treatment and reached maximum after 48 h (Fig. 5).

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