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Selection and validation of reference genes for quantitative gene expression studies by real-time PCR in eggplant (Solanum melongena L).

Gantasala NP, Papolu PK, Thakur PK, Kamaraju D, Sreevathsa R, Rao U - BMC Res Notes (2013)

Bottom Line: The candidate genes were cloned from cDNA and analysed by real-time PCR.The expression data analyzed by three statistical methods (geNorm, NormFinder and BestKeeper) identified 18s rRNA, Cyclophilin and APRT as the most stable and suitable reference genes in eggplant.This was further confirmed in four different varieties, two representative lines of transgenic eggplant as well as in nematode infected eggplant. 18s rRNA, Cyclophilin and APRT have been found to be appropriate for the normalization of real-time PCR data for gene expression studies in eggplant.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Nematology, Indian Agricultural Research Institute, New Delhi 110012, India.

ABSTRACT

Background: Analysis of gene expression patterns leads to functional understanding of biological processes. Quantitative real-time PCR has become the most commonly used technique for in-depth studies of gene expression. To quantify variation in specific gene expression, accurate and reliable normalization across different samples and tissues is necessary. This can be achieved by selecting one or more suitable reference genes to compare the target mRNA transcript levels. In the present work, we illustrate the first evaluation of potential internal control or reference genes across different developmental stages of eggplant for reliable quantification of transcripts by real-time PCR.

Results: We have evaluated the stability in expression of six candidate reference genes (18s rRNA, APRT, GAPDH, Cyclophilin, Actin, and RuBP) in a set of tissues representing six developmental stages of eggplant. The candidate genes were cloned from cDNA and analysed by real-time PCR. The expression data analyzed by three statistical methods (geNorm, NormFinder and BestKeeper) identified 18s rRNA, Cyclophilin and APRT as the most stable and suitable reference genes in eggplant. This was further confirmed in four different varieties, two representative lines of transgenic eggplant as well as in nematode infected eggplant.

Conclusion: 18s rRNA, Cyclophilin and APRT have been found to be appropriate for the normalization of real-time PCR data for gene expression studies in eggplant.

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Specificity of real-time PCR amplification (A) Quantification curve for the six candidate reference genes from cDNA of six different tissues (B) Melting curve for the six candidate reference genes from cDNA of six different.
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Figure 3: Specificity of real-time PCR amplification (A) Quantification curve for the six candidate reference genes from cDNA of six different tissues (B) Melting curve for the six candidate reference genes from cDNA of six different.

Mentions: Genes encoding for 18s rRNA, adenine phosphoribosyl transferase (APRT), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Cyclophilin, Actin, Ribulose-1,5-bisphosphate carboxylase (RuBP) were selected based on previous studies that relied on them as candidate reference genes [23-25]. In order to calculate the stability of expression of the selected six candidate reference genes, mRNA expression levels were measured in six different tissues of the eggplant (young leaf, mature leaf, shoot, root, flower bud and open flower). Ct mean values of three biological replicates were obtained from Realplex2 software. These Ct mean values were further used for the calculation of expression stability (Figure 3A &3B, Table 2). Real-time PCR analysis revealed a large significant similarity in the observed expression pattern of all the genes across various tissues. Three commonly used statistical algorithms viz., BestKeeper, Normfinder and geNorm were employed for normalization of expression pattern and to validate Ct values for choosing the best reference genes.


Selection and validation of reference genes for quantitative gene expression studies by real-time PCR in eggplant (Solanum melongena L).

Gantasala NP, Papolu PK, Thakur PK, Kamaraju D, Sreevathsa R, Rao U - BMC Res Notes (2013)

Specificity of real-time PCR amplification (A) Quantification curve for the six candidate reference genes from cDNA of six different tissues (B) Melting curve for the six candidate reference genes from cDNA of six different.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750715&req=5

Figure 3: Specificity of real-time PCR amplification (A) Quantification curve for the six candidate reference genes from cDNA of six different tissues (B) Melting curve for the six candidate reference genes from cDNA of six different.
Mentions: Genes encoding for 18s rRNA, adenine phosphoribosyl transferase (APRT), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Cyclophilin, Actin, Ribulose-1,5-bisphosphate carboxylase (RuBP) were selected based on previous studies that relied on them as candidate reference genes [23-25]. In order to calculate the stability of expression of the selected six candidate reference genes, mRNA expression levels were measured in six different tissues of the eggplant (young leaf, mature leaf, shoot, root, flower bud and open flower). Ct mean values of three biological replicates were obtained from Realplex2 software. These Ct mean values were further used for the calculation of expression stability (Figure 3A &3B, Table 2). Real-time PCR analysis revealed a large significant similarity in the observed expression pattern of all the genes across various tissues. Three commonly used statistical algorithms viz., BestKeeper, Normfinder and geNorm were employed for normalization of expression pattern and to validate Ct values for choosing the best reference genes.

Bottom Line: The candidate genes were cloned from cDNA and analysed by real-time PCR.The expression data analyzed by three statistical methods (geNorm, NormFinder and BestKeeper) identified 18s rRNA, Cyclophilin and APRT as the most stable and suitable reference genes in eggplant.This was further confirmed in four different varieties, two representative lines of transgenic eggplant as well as in nematode infected eggplant. 18s rRNA, Cyclophilin and APRT have been found to be appropriate for the normalization of real-time PCR data for gene expression studies in eggplant.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Nematology, Indian Agricultural Research Institute, New Delhi 110012, India.

ABSTRACT

Background: Analysis of gene expression patterns leads to functional understanding of biological processes. Quantitative real-time PCR has become the most commonly used technique for in-depth studies of gene expression. To quantify variation in specific gene expression, accurate and reliable normalization across different samples and tissues is necessary. This can be achieved by selecting one or more suitable reference genes to compare the target mRNA transcript levels. In the present work, we illustrate the first evaluation of potential internal control or reference genes across different developmental stages of eggplant for reliable quantification of transcripts by real-time PCR.

Results: We have evaluated the stability in expression of six candidate reference genes (18s rRNA, APRT, GAPDH, Cyclophilin, Actin, and RuBP) in a set of tissues representing six developmental stages of eggplant. The candidate genes were cloned from cDNA and analysed by real-time PCR. The expression data analyzed by three statistical methods (geNorm, NormFinder and BestKeeper) identified 18s rRNA, Cyclophilin and APRT as the most stable and suitable reference genes in eggplant. This was further confirmed in four different varieties, two representative lines of transgenic eggplant as well as in nematode infected eggplant.

Conclusion: 18s rRNA, Cyclophilin and APRT have been found to be appropriate for the normalization of real-time PCR data for gene expression studies in eggplant.

Show MeSH