Reproducibility, bioinformatic analysis and power of the SAGE method to evaluate changes in transcriptome - PubMed (original) (raw)
Reproducibility, bioinformatic analysis and power of the SAGE method to evaluate changes in transcriptome
S Dinel et al. Nucleic Acids Res. 2005.
Abstract
The serial analysis of gene expression (SAGE) method is used to study global gene expression in cells or tissues in various experimental conditions. However, its reproducibility has not yet been definitively assessed. In this study, we have evaluated the reproducibility of the SAGE method and identified the factors that affect it. The determination coefficient (R2 ) for the reproducibility of SAGE is 0.96. However, there are some factors that can affect the reproducibility of SAGE, such as the replication of concatemers and ditags, the number of sequenced tags and double PCR amplification of ditags. Thus, corrections for these factors must be made to ensure the reproducibility and accuracy of SAGE results. A bioinformatic analysis of SAGE data is also presented in order to eliminate these artifacts. Finally, the current study shows that increasing the number of sequenced tags improves the power of the method to detect transcripts and their regulation by experimental conditions.
Figures
Figure 1
Comparison of individual tag abundance estimated from two SAGE libraries of about 50 000 (a) or 150 000 (b) tags, each independently generated from the same pool of total RNA.
Figure 2
Overview of the SAGEparser software package.
Figure 3
Reproducibility of the SAGE method with one or two PCR amplifications.
Figure 4
Comparison of the results obtained by microarrays and (a) the SAGE method or (b) the SADE method with two PCR amplifications.
Figure 5
(a) Influence of the number of sequenced tags on the detection of transcript species. (b) Type of transcript species detected according to the number of tags sequenced.
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