Limits...
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.

Show MeSH

Related in: MedlinePlus

Expression stability and ranking of the candidate reference genes by geNorm.Expression stability and ranking of 11 reference genes calculated with geNorm in all the samples (A), Wounding (B), Salt (C), Drought (D), Heat (E), Cold (F), Tissue (G), MJ (H), SA (I), ABA (J) and Biotic (K). A lower M value indicates more stable gene expression.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4359125&req=5

pone.0118860.g003: Expression stability and ranking of the candidate reference genes by geNorm.Expression stability and ranking of 11 reference genes calculated with geNorm in all the samples (A), Wounding (B), Salt (C), Drought (D), Heat (E), Cold (F), Tissue (G), MJ (H), SA (I), ABA (J) and Biotic (K). A lower M value indicates more stable gene expression.

Mentions: The expression stability of candidate reference genes under different stress conditions was evaluated by using different approaches. Relative quantities of all samples were exported to geNorm (version 3.5) to analyze gene expression stability. Stability analysis by geNorm is based on the M value, which is calculated by stepwise exclusion of the unstable genes followed by recalculation of M values [27]. M value less than the threshold value of 1.5 has been recommended by Vandesompele et al [27]. In our analysis only 3 genes showed M value under the recommended value of 1.5 and rest of the five genes were above this value. It was observed that the stability of the genes varied according with different treatments and most of them showed inconsistently under all experimental conditions. Starting from the two most stable genes on the right (lowest M value), the genes are ranked according to decreasing expression stability, ending with the most unstable genes on the left with highest M value. The most stable reference genes were not identical in all individual stress treated samples. TUB and RPL2 (0.39) seems to be the most stable genes in salt treated samples, meanwhile RPL2 and 26S (0.21) in drought treatment, and CYP and EF-1 (1.15) in heat treated samples. Similarly, among other 11 genes CYP and UBQ were found to be most stable genes with M value of 0.30 for wounding and cold treatment. CYP, TUB (0.45) for MJ samples and T-SAND, UBQ (0.52) for SA treated samples showed lowest M values, thus were most stable genes. For ABA treatment RPL2 and EF-1 (0.64) exhibited stable expression as compare to other genes. Under biotic stress UBQ and T-SAND genes were on the top with lowest M value of 0.021. For all combined treatments, different tissues and heat treatment CYP and EF-1 genes were found to be most stable (Fig. 3). Rest of the genes were found to be unstable with higher M values than cut off value of 1.5. Among all the reference genes examined 18S-rRNA and TUA were found to be the least stable with highest M value.


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)

Expression stability and ranking of the candidate reference genes by geNorm.Expression stability and ranking of 11 reference genes calculated with geNorm in all the samples (A), Wounding (B), Salt (C), Drought (D), Heat (E), Cold (F), Tissue (G), MJ (H), SA (I), ABA (J) and Biotic (K). A lower M value indicates more stable gene expression.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118860.g003: Expression stability and ranking of the candidate reference genes by geNorm.Expression stability and ranking of 11 reference genes calculated with geNorm in all the samples (A), Wounding (B), Salt (C), Drought (D), Heat (E), Cold (F), Tissue (G), MJ (H), SA (I), ABA (J) and Biotic (K). A lower M value indicates more stable gene expression.
Mentions: The expression stability of candidate reference genes under different stress conditions was evaluated by using different approaches. Relative quantities of all samples were exported to geNorm (version 3.5) to analyze gene expression stability. Stability analysis by geNorm is based on the M value, which is calculated by stepwise exclusion of the unstable genes followed by recalculation of M values [27]. M value less than the threshold value of 1.5 has been recommended by Vandesompele et al [27]. In our analysis only 3 genes showed M value under the recommended value of 1.5 and rest of the five genes were above this value. It was observed that the stability of the genes varied according with different treatments and most of them showed inconsistently under all experimental conditions. Starting from the two most stable genes on the right (lowest M value), the genes are ranked according to decreasing expression stability, ending with the most unstable genes on the left with highest M value. The most stable reference genes were not identical in all individual stress treated samples. TUB and RPL2 (0.39) seems to be the most stable genes in salt treated samples, meanwhile RPL2 and 26S (0.21) in drought treatment, and CYP and EF-1 (1.15) in heat treated samples. Similarly, among other 11 genes CYP and UBQ were found to be most stable genes with M value of 0.30 for wounding and cold treatment. CYP, TUB (0.45) for MJ samples and T-SAND, UBQ (0.52) for SA treated samples showed lowest M values, thus were most stable genes. For ABA treatment RPL2 and EF-1 (0.64) exhibited stable expression as compare to other genes. Under biotic stress UBQ and T-SAND genes were on the top with lowest M value of 0.021. For all combined treatments, different tissues and heat treatment CYP and EF-1 genes were found to be most stable (Fig. 3). Rest of the genes were found to be unstable with higher M values than cut off value of 1.5. Among all the reference genes examined 18S-rRNA and TUA were found to be the least stable with highest M value.

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