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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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Multiple Sequence Alignment of the six candidate genes by CLUSTALW.
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Figure 2: Multiple Sequence Alignment of the six candidate genes by CLUSTALW.

Mentions: PCR amplification of six target reference genes viz., 18s rRNA, APRT, GAPDH, Cyclophilin, Actin, RuBP from the cDNA resulted in 416 bp, 454 bp, 586 bp, 265 bp, 333 bp, and 269 bp amplicons respectively, which were cloned and sequenced (Figure 1A &1B). Moreover comparative analysis of the sequenced products revealed high (95-100%) similarity with the members of Solanaceae family (Figure 2 &Table 1). The sequences were later deposited in the GenBank database (GenBank: JX448341, JX448342, JX448343, JX448344, JX448345, JX524155). In order to check the presence of introns between the primer binding sites, all the six reference genes were PCR amplified from the genomic DNA extracted from the eggplant leaves (Figure 1C). Except for APRT, amplification was successful for all the other five genes. There was no amplification in APRT probably due to the presence of a big intronic region; in case of GAPDH, the amplicon was bigger than the cDNA fragment indicating the presence of an intron. With respect to the other four genes, the size of the genomic DNA fragments was same as that of the cDNA fragments.


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)

Multiple Sequence Alignment of the six candidate genes by CLUSTALW.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Multiple Sequence Alignment of the six candidate genes by CLUSTALW.
Mentions: PCR amplification of six target reference genes viz., 18s rRNA, APRT, GAPDH, Cyclophilin, Actin, RuBP from the cDNA resulted in 416 bp, 454 bp, 586 bp, 265 bp, 333 bp, and 269 bp amplicons respectively, which were cloned and sequenced (Figure 1A &1B). Moreover comparative analysis of the sequenced products revealed high (95-100%) similarity with the members of Solanaceae family (Figure 2 &Table 1). The sequences were later deposited in the GenBank database (GenBank: JX448341, JX448342, JX448343, JX448344, JX448345, JX524155). In order to check the presence of introns between the primer binding sites, all the six reference genes were PCR amplified from the genomic DNA extracted from the eggplant leaves (Figure 1C). Except for APRT, amplification was successful for all the other five genes. There was no amplification in APRT probably due to the presence of a big intronic region; in case of GAPDH, the amplicon was bigger than the cDNA fragment indicating the presence of an intron. With respect to the other four genes, the size of the genomic DNA fragments was same as that of the cDNA fragments.

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