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Serial Analysis of Gene Expression (SAGE): Digital Gene Expression Profiling facilitates the introduction of SAGE into the laboratory, and provides a framework for interpreting and comparing data derived from SAGE experiments. Of the several methods of genetic profiling available, only SAGE measures the expression of both known and unknown genes. SAGE studies encompass 50,000 tags and can provide detailed knowledge of the 2000 most highly expressed genes in the tissue sample. The SAGE protocols presented are detailed, fully annotated, and tested, and are all written by experienced SAGE researchers from around the world. Part 1 is dedicated to experimental procedures of SAGE and related methods including aRNA LongSAGE, SuperSAGE, DeepSAGE, and GMAT. Part 2 provides methods for extraction and filtration of tags, analysis of ditag populations, and completing statistically correct comparisons of gene expression profiles.

 

Comparative transcriptomics enables scientists to understand the underlying genetics of biological changes such as development, disease, crop yield, and resistance. SAGE analysis is also used to obtain unknown tags, which can be used as gene-specific primers in Rapid Amplification of cDNA Ends (RACE) reactions to generate full-length transcripts for cloning and sequencing. This book will be an indispensable tool for any lab engaged in genetic profiling and comparative transcriptomics, and will help many laboratories to successfully implement tag-based sequencing methods and procedures and obtain comprehensive, useful, and interpretative data.

Table of contents

Contents.

 

Preface

Contributors

 

Part 1: Experimental Procedures

 

1. SAGE and LongSAGE.

Annabeth Laursen Høgh and Kåre Lehmann Nielsen.

 

2. Robust-LongSAGE (RL-SAGE): an improved LongSAGE method for high-throughput transcriptome analysis.

Malali Gowda and Guo-Liang Wang.

 

3. aRNALongsAGE: SAGE with Antisense RNA.

Anna M. Heidenblut.

 

4. SuperSAGE

Hideo Matsumura, Monika Reuter, Detlev H. Krüger, Peter Winter, Günter Kahl and Ryohei Terauchi.

 

5. Low Cost Medium Throughput Sanger Dideoxy-Sequencing.

Kåre Lehmann Nielsen.

 

6. DeepSAGE-Higher Sensitivity and Multiplexing of Samples Using a Simpler Experimental Protocol.

Kåre Lehmann Nielsen.

 

7. High-Resolution Genome-wide Mapping of Chromatin Modifications by GMAT

Tae-Young Roh and Keji Zhao.

 

8. 5'-and 3'-RACE from LongSAGE tags.

Kåre Lehmann Nielsen.

 

Part 2: Tag Extraction and Analysis.

 

9. Extraction and Annotation of SAGE tags using Sequence Quality values.

Jeppe Emmersen.

 

10. Correction of technology-Related artifacts in Serial Analysis of Gene Expression.

Viatcheslav R. Akmaev.

 

11. Duplicate Ditag analysis in LongSAGE

Jeppe Emmersen.

 

12. Statistical Comparison of Two or More SAGE Libraries: One Tag at the Time.

Gerben J. Schaaf, Fred van Ruissen, Antoine van Kampen, Marcel Kool and Jan M. Ruijter.

 

13. Scaling of Gene Expression Data Allowing the Comparison of Different Gene Expression Platforms.

Fred van Ruissen, Gerben J. Schaaf, Marcel Kool, Frank Baas and Jan M. Ruijter.

 

14. Clustering Analysis of SAGE Transcription Profiles Using a Poisson Approach.

Haiyan Huang, Li Cai and Wing H. Wong.

 

15. Identifying Non-specific SAGE tags by Context of Gene Expression.

Xijin Ge and San Ming Wang.