Limits...
Ratio-based analysis of differential mRNA processing and expression of a polyadenylation factor mutant pcfs4 using arabidopsis tiling microarray.

Zheng J, Xing D, Wu X, Shen Y, Kroll DM, Ji G, Li QQ - PLoS ONE (2011)

Bottom Line: Quantitative PCR analysis of a set of DPGs confirmed that most of these genes were truly differentially processed in pcfs4 mutant plants.The enriched GO term "regulation of flower development" among PCFS4 targets further indicated the efficacy of the RADPRE pipeline.This simple but effective program is available upon request.

View Article: PubMed Central - PubMed

Affiliation: Department of Automation, Xiamen University, Xiamen, Fujian, China.

ABSTRACT

Background: Alternative polyadenylation as a mechanism in gene expression regulation has been widely recognized in recent years. Arabidopsis polyadenylation factor PCFS4 was shown to function in leaf development and in flowering time control. The function of PCFS4 in controlling flowering time was correlated with the alternative polyadenylation of FCA, a flowering time regulator. However, genetic evidence suggested additional targets of PCFS4 that may mediate its function in both flowering time and leaf development.

Methodology/principal findings: To identify further targets, we investigated the whole transcriptome of a PCFS4 mutant using Affymetrix Arabidopsis genomic tiling 1.0R array and developed a data analysis pipeline, termed RADPRE (Ratio-based Analysis of Differential mRNA Processing and Expression). In RADPRE, ratios of normalized probe intensities between wild type Columbia and a pcfs4 mutant were first generated. By doing so, one of the major problems of tiling array data--variations caused by differential probe affinity--was significantly alleviated. With the probe ratios as inputs, a hierarchy of statistical tests was carried out to identify differentially processed genes (DPG) and differentially expressed genes (DEG). The false discovery rate (FDR) of this analysis was estimated by using the balanced random combinations of Col/pcfs4 and pcfs4/Col ratios as inputs. Gene Ontology (GO) analysis of the DPGs and DEGs revealed potential new roles of PCFS4 in stress responses besides flowering time regulation.

Conclusion/significance: We identified 68 DPGs and 114 DEGs with FDR at 1% and 2%, respectively. Most of the 68 DPGs were subjected to alternative polyadenylation, splicing or transcription initiation. Quantitative PCR analysis of a set of DPGs confirmed that most of these genes were truly differentially processed in pcfs4 mutant plants. The enriched GO term "regulation of flower development" among PCFS4 targets further indicated the efficacy of the RADPRE pipeline. This simple but effective program is available upon request.

Show MeSH
Box plots of log-transformed raw data (left panel), VSN-normalized (middle panel) and RMA-normalized (right panel) data.The across-array variations were significantly reduced after normalization.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3045369&req=5

pone-0014719-g002: Box plots of log-transformed raw data (left panel), VSN-normalized (middle panel) and RMA-normalized (right panel) data.The across-array variations were significantly reduced after normalization.

Mentions: The quality of the array data is essential for extracting biologically meaningful information. Therefore, the six CEL file data were first checked for their quality through “exploratory” data analysis with raw and log-transformed intensities [39]. The even distributions of signal intensities across the chips indicated high quality original data (Figure S1). Among the available methods for background correction and across-array normalization, “VSN” and “RMA” were chosen for the processing of our array data [38], [40]. The box-plots of before- and after-normalization data clearly indicated that the normalization removed the across-array bias and rendered the data distribution more consistent among the 6 data files (Figure 2). Although there was no significant difference observed between VSN and RMA normalizations, the signal intensities were less varied with the former method (Figure 2). Therefore, the down-stream data analysis was carried out using the “VSN” normalized data.


Ratio-based analysis of differential mRNA processing and expression of a polyadenylation factor mutant pcfs4 using arabidopsis tiling microarray.

Zheng J, Xing D, Wu X, Shen Y, Kroll DM, Ji G, Li QQ - PLoS ONE (2011)

Box plots of log-transformed raw data (left panel), VSN-normalized (middle panel) and RMA-normalized (right panel) data.The across-array variations were significantly reduced after normalization.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045369&req=5

pone-0014719-g002: Box plots of log-transformed raw data (left panel), VSN-normalized (middle panel) and RMA-normalized (right panel) data.The across-array variations were significantly reduced after normalization.
Mentions: The quality of the array data is essential for extracting biologically meaningful information. Therefore, the six CEL file data were first checked for their quality through “exploratory” data analysis with raw and log-transformed intensities [39]. The even distributions of signal intensities across the chips indicated high quality original data (Figure S1). Among the available methods for background correction and across-array normalization, “VSN” and “RMA” were chosen for the processing of our array data [38], [40]. The box-plots of before- and after-normalization data clearly indicated that the normalization removed the across-array bias and rendered the data distribution more consistent among the 6 data files (Figure 2). Although there was no significant difference observed between VSN and RMA normalizations, the signal intensities were less varied with the former method (Figure 2). Therefore, the down-stream data analysis was carried out using the “VSN” normalized data.

Bottom Line: Quantitative PCR analysis of a set of DPGs confirmed that most of these genes were truly differentially processed in pcfs4 mutant plants.The enriched GO term "regulation of flower development" among PCFS4 targets further indicated the efficacy of the RADPRE pipeline.This simple but effective program is available upon request.

View Article: PubMed Central - PubMed

Affiliation: Department of Automation, Xiamen University, Xiamen, Fujian, China.

ABSTRACT

Background: Alternative polyadenylation as a mechanism in gene expression regulation has been widely recognized in recent years. Arabidopsis polyadenylation factor PCFS4 was shown to function in leaf development and in flowering time control. The function of PCFS4 in controlling flowering time was correlated with the alternative polyadenylation of FCA, a flowering time regulator. However, genetic evidence suggested additional targets of PCFS4 that may mediate its function in both flowering time and leaf development.

Methodology/principal findings: To identify further targets, we investigated the whole transcriptome of a PCFS4 mutant using Affymetrix Arabidopsis genomic tiling 1.0R array and developed a data analysis pipeline, termed RADPRE (Ratio-based Analysis of Differential mRNA Processing and Expression). In RADPRE, ratios of normalized probe intensities between wild type Columbia and a pcfs4 mutant were first generated. By doing so, one of the major problems of tiling array data--variations caused by differential probe affinity--was significantly alleviated. With the probe ratios as inputs, a hierarchy of statistical tests was carried out to identify differentially processed genes (DPG) and differentially expressed genes (DEG). The false discovery rate (FDR) of this analysis was estimated by using the balanced random combinations of Col/pcfs4 and pcfs4/Col ratios as inputs. Gene Ontology (GO) analysis of the DPGs and DEGs revealed potential new roles of PCFS4 in stress responses besides flowering time regulation.

Conclusion/significance: We identified 68 DPGs and 114 DEGs with FDR at 1% and 2%, respectively. Most of the 68 DPGs were subjected to alternative polyadenylation, splicing or transcription initiation. Quantitative PCR analysis of a set of DPGs confirmed that most of these genes were truly differentially processed in pcfs4 mutant plants. The enriched GO term "regulation of flower development" among PCFS4 targets further indicated the efficacy of the RADPRE pipeline. This simple but effective program is available upon request.

Show MeSH