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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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Expression stability and ranking of housekeeping genes by three statistical softwares, (A) Best keeper (B) NormFinder (C) geNorm.
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Figure 4: Expression stability and ranking of housekeeping genes by three statistical softwares, (A) Best keeper (B) NormFinder (C) geNorm.

Mentions: This analysis was done using the raw Ct values. Initially, variations (SD (± Ct) and CV (%Ct)) were calculated for each of the candidate reference genes in the samples for identifying the overall stability in gene expression. Three candidate reference genes (GAPDH, Actin and RuBP) showed an SD value higher than 1, which disqualified their utility as reference genes. Remaining three genes were selected for further analysis as they showed SD value less than 1. Further processing of the data using pair wise correlation and regression analysis showed the inter gene relations and eliminated APRT, as the gene with the least correlation (r = 0.481) (Table 3). The analysis of the remaining two genes (18s rRNA and Cyclophilin) showed a strong and significant correlation with an ‘r’ value of 0.990 for 18S rRNA and 0.895 for Cyclophilin indicating their stable expression levels. Accordingly, the BestKeeper index (p) was found to be 0.001 and 0.016 respectively for 18s rRNA and Cyclophilin (Figure 4A). In view of high correlation value and low BestKeeper index, 18s rRNA and Cyclophilin were selected as the two likely 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)

Expression stability and ranking of housekeeping genes by three statistical softwares, (A) Best keeper (B) NormFinder (C) geNorm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Expression stability and ranking of housekeeping genes by three statistical softwares, (A) Best keeper (B) NormFinder (C) geNorm.
Mentions: This analysis was done using the raw Ct values. Initially, variations (SD (± Ct) and CV (%Ct)) were calculated for each of the candidate reference genes in the samples for identifying the overall stability in gene expression. Three candidate reference genes (GAPDH, Actin and RuBP) showed an SD value higher than 1, which disqualified their utility as reference genes. Remaining three genes were selected for further analysis as they showed SD value less than 1. Further processing of the data using pair wise correlation and regression analysis showed the inter gene relations and eliminated APRT, as the gene with the least correlation (r = 0.481) (Table 3). The analysis of the remaining two genes (18s rRNA and Cyclophilin) showed a strong and significant correlation with an ‘r’ value of 0.990 for 18S rRNA and 0.895 for Cyclophilin indicating their stable expression levels. Accordingly, the BestKeeper index (p) was found to be 0.001 and 0.016 respectively for 18s rRNA and Cyclophilin (Figure 4A). In view of high correlation value and low BestKeeper index, 18s rRNA and Cyclophilin were selected as the two likely 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