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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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Distribution of transcript populations of 11 selected genes under different stress treatments.In each treatment, the transcript amount of each gene is shown as a ratio relative to the sum of the 11 transcript populations. The samples were collected at 0hr, 6hr, 12hr, 24hr and 48hr interval of time after each treatment. Different treatments were heat, cold, drought, wounding, salt, MJ, SA, ABA, biotic and tissue (Flower, leaf, stem root and whole plant).
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pone.0118860.g002: Distribution of transcript populations of 11 selected genes under different stress treatments.In each treatment, the transcript amount of each gene is shown as a ratio relative to the sum of the 11 transcript populations. The samples were collected at 0hr, 6hr, 12hr, 24hr and 48hr interval of time after each treatment. Different treatments were heat, cold, drought, wounding, salt, MJ, SA, ABA, biotic and tissue (Flower, leaf, stem root and whole plant).

Mentions: Total RNA extracted from leaf samples was evaluated for quality and integrity. RNA integrity was checked on agarose gel electrophoresis (S1 Fig.). Real-time qRT-PCR was conducted with 11 candidate reference genes; 18S ribosomal RNA (18S-rRNA), 26S ribosomal RNA (26S), Actin (ACT), cyclophylin (CYP), elongation factor-1 (EF-1), glyceral-dehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L2 (RPL2), Sand family protein (SAND), alpha-tubulin (TUA), beta-tubulin (TUB) and ubiquitin (UBQ). The specificity of the designed primer pairs was assessed by a confirmatory PCR and 2% agarose gel electrophoresis that revealed expected single bands of desired lengths (S2 Fig.). Further, the PCR amplify fragments were sequenced to validate specificity of gene primers. Furthermore, specificity was also confirmed from single peak in dissociation curve and without peak in no template control (NTC) which revealed the absence of any genomic DNA (S3 Fig.). PCR efficiency (E) and correlation coefficient R2 was estimated from standard curve showed range between 86–102% and 0.991–1.00, respectively (Table 1). The Cq values of the raw expression data across all samples are shown in Fig. 1, which varied from 7.44 to 36.48 and mostly lying between 19–23 range. Among all tested reference genes, 18S-rRNA showed the highest abundance (lowest Cq value) with a mean of 10.28 followed by EF-1 (21.11) and TUB (21.20), CYP (22.06), TUA (22.66), UBQ (23.59), 26S (28.48), GAPDH (29.19). RPL2, T-SAND and ACT showed the least transcript abundance with a mean Cq of 33.65, 31.83 and 31.79, respectively (Fig. 1). UBQ, RPL2, 18S-rRNA and CYP showed the lowest gene expression variation (below 4 cycles) while TUA, TUB and 26S had highest expression variation (above 7 cycles). Transcript quantities are shown for each treatment as ratios relative to the sum of the all transcript populations. Variations in the relative quantities of the transcripts with different treatments are shown in Fig. 2. Such type variation in the expression level reflects that there is lack of consistency in their expression pattern under experimental stress responses. For example 18S-rRNA mRNA represents approximately 10% of the total mRNA population in wounding treated samples but more than 40% in heat treatment. We found that the amount of mRNA transcript of all genes varied in all treatment. The transcript level of CYP remained relatively constant in wounding, different plant organs and biotic treatment. Similarly transcript level of T-SAND remained constant for salt and salicylic acid treatment and those of RPL2 were also remained constant for drought treated samples. Relative transcript level of T-SAND varied in all samples but maximum level was observed in heat treatment. From these results it can be concluded that expression level of most of the genes is not constant, it varies with different treatments. So there is need to analyze the expression stability of each gene.


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)

Distribution of transcript populations of 11 selected genes under different stress treatments.In each treatment, the transcript amount of each gene is shown as a ratio relative to the sum of the 11 transcript populations. The samples were collected at 0hr, 6hr, 12hr, 24hr and 48hr interval of time after each treatment. Different treatments were heat, cold, drought, wounding, salt, MJ, SA, ABA, biotic and tissue (Flower, leaf, stem root and whole plant).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118860.g002: Distribution of transcript populations of 11 selected genes under different stress treatments.In each treatment, the transcript amount of each gene is shown as a ratio relative to the sum of the 11 transcript populations. The samples were collected at 0hr, 6hr, 12hr, 24hr and 48hr interval of time after each treatment. Different treatments were heat, cold, drought, wounding, salt, MJ, SA, ABA, biotic and tissue (Flower, leaf, stem root and whole plant).
Mentions: Total RNA extracted from leaf samples was evaluated for quality and integrity. RNA integrity was checked on agarose gel electrophoresis (S1 Fig.). Real-time qRT-PCR was conducted with 11 candidate reference genes; 18S ribosomal RNA (18S-rRNA), 26S ribosomal RNA (26S), Actin (ACT), cyclophylin (CYP), elongation factor-1 (EF-1), glyceral-dehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L2 (RPL2), Sand family protein (SAND), alpha-tubulin (TUA), beta-tubulin (TUB) and ubiquitin (UBQ). The specificity of the designed primer pairs was assessed by a confirmatory PCR and 2% agarose gel electrophoresis that revealed expected single bands of desired lengths (S2 Fig.). Further, the PCR amplify fragments were sequenced to validate specificity of gene primers. Furthermore, specificity was also confirmed from single peak in dissociation curve and without peak in no template control (NTC) which revealed the absence of any genomic DNA (S3 Fig.). PCR efficiency (E) and correlation coefficient R2 was estimated from standard curve showed range between 86–102% and 0.991–1.00, respectively (Table 1). The Cq values of the raw expression data across all samples are shown in Fig. 1, which varied from 7.44 to 36.48 and mostly lying between 19–23 range. Among all tested reference genes, 18S-rRNA showed the highest abundance (lowest Cq value) with a mean of 10.28 followed by EF-1 (21.11) and TUB (21.20), CYP (22.06), TUA (22.66), UBQ (23.59), 26S (28.48), GAPDH (29.19). RPL2, T-SAND and ACT showed the least transcript abundance with a mean Cq of 33.65, 31.83 and 31.79, respectively (Fig. 1). UBQ, RPL2, 18S-rRNA and CYP showed the lowest gene expression variation (below 4 cycles) while TUA, TUB and 26S had highest expression variation (above 7 cycles). Transcript quantities are shown for each treatment as ratios relative to the sum of the all transcript populations. Variations in the relative quantities of the transcripts with different treatments are shown in Fig. 2. Such type variation in the expression level reflects that there is lack of consistency in their expression pattern under experimental stress responses. For example 18S-rRNA mRNA represents approximately 10% of the total mRNA population in wounding treated samples but more than 40% in heat treatment. We found that the amount of mRNA transcript of all genes varied in all treatment. The transcript level of CYP remained relatively constant in wounding, different plant organs and biotic treatment. Similarly transcript level of T-SAND remained constant for salt and salicylic acid treatment and those of RPL2 were also remained constant for drought treated samples. Relative transcript level of T-SAND varied in all samples but maximum level was observed in heat treatment. From these results it can be concluded that expression level of most of the genes is not constant, it varies with different treatments. So there is need to analyze the expression stability of each gene.

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