If theme and variation of a technical principle is the ultimate form of tribute to its utility, then the modifications of the serial analysis of gene expression (SAGE) technique published to date establish it as a powerful molecular tool invented in the genome era. As described in this opus cataloguing the refinements, modifications and applications of this methodology, the SAGE technique has reached a level of maturity and complexity that justify a review book.
The growing list of acronyms of modified techniques are formidable and include: longSAGE, SuperSAGE, 5´ SAGE, CAGE, TEC-RED, SARA-PCR, GIS and GLGI for transcriptional analyses and; SACO, STAGE, GMAT and DACS for genomic analyses. SAGE: Current Technologies and Applications (Horizon Bioscience, Norfolk, UK, 2005), edited by San Ming Wang, who has also contributed to its advancement (Chapter 18), is a comprehensive collection of chapters by experts from disparate fields, such as bioinformatics and plant biology, with their individual interests, who detail their latest progress in the refinement and use of this genetic technique.
First reported in Science in 1995 in parallel with the microarray gene expression method, the SAGE technique has undergone various optimizations and modifications, and has been applied by investigators around the world. It is based on two principles: a short cDNA sequence tag at a fixed distance from the polyA tail of a transcript is of sufficient complexity to uniquely identify the gene from which the transcript originated; and the serial concatenation of tags enables the efficient sequencing and determination of transcript frequency. Demonstrating the widespread use of this technology, there are now reportedly over 15 million human SAGE tags available on the Gene Expression Omnibus repository contributed by various sources, both independent investigators and collaborative efforts, such as the Cancer Genome Anatomy Project.
The first nine chapters of this book are technically oriented, some of which are quite instructional with useful detailed protocols. The first chapter appropriately starts with an excellent introduction and expounds one of the two important principles stated above: identifying the SAGE tag gene of origin. Although this is a rather simple concept on first pass, it is rigorously examined using transcriptomes of model organisms such as Caenorhabditis elegans and Drosophila melanogaster to propose a tag length with maximal utility. Potential errors and biases that may occur during SAGE library construction, such as incomplete enzyme digestion, linker contamination, differences in the efficiency of ditag ligation and PCR amplification due to tag sequence, are also discussed along with solutions. The additional benefit of creating longer 26-bp tags (SuperSAGE, Chapter 3) for potential use in RNA interference and clarifying gene function is discussed.
Modifications of the SAGE protocol to help identify transcripts are presented in subsequent chapters. CAGE (cap analysis gene expression; Chapter 2) focuses on identifying the 5´ end of the transcript along with potential promoter elements. For improving tag-to-gene assignment at the 3´ end of the transcript, SARA (SAGE rapid amplification of cDNA ends)-PCR and an adaptation of the GLGI method (generation of longer cDNA fragments from SAGE tags for gene identification) are described in Chapters 4 and 7, respectively. A short Chapter 5 describes the purification of ditags by reverse-phase, high-performance liquid chromatography rather than polyacrylamide gel electrophoresis for SAGE library construction. Although anomalously placed in the midst of tag-to-gene identification chapters, it may hold the answer for those seeking to improve concatemerization reactions, a critical step for a high-quality SAGE library.
With the seemingly geometric expansion of genomic information, knowing how to find, analyze and compare these data is important for advancing our knowledge regarding the biology of the studied systems. Although there is some overlap in subject coverage amongst the chapters, the three that are devoted to web tools and SAGE data analyses are useful reviews, even for those who are familiar with the technique. The conceptual discussions, references, resource links and tools in Chapters 6 are good starting points for accessing the invaluable SAGE database, whether or not the reader is actually planning to perform SAGE experiments. Dedicated websites have been created by the Johns Hopkins Oncology Center (SAGENet), National Center of Biotechnology Information (SAGEmap) and Cancer Genome Anatomy Project (SAGE Genie) by the National Cancer Institute. For in-depth data analysis and theoretical considerations, Chapters 8 and 9 appear to engage even the most mathematically inclined reader.
The SAGE technique has been used in an estimated 1000 publications by report, one-half using human and one-third using rodent tissues. The majority of studies have been related to cancer biology as may be expected by its invention in a molecular oncology laboratory and earlier application by cancer biologists. Nonetheless, the second half of the book gives a more balanced coverage of its application in diverse fields. It covers a broad range of areas, such as plant (rice, Arabidopsis and loblolly pine, Chapters 10 and 16), insect (Drosophila, Chapter 15) and embryonic stem cell systems (Chapter 11). The effects of biological states, such as exercise (Chapter 14), age and gender differences (Chapter 17), on gene expression are examined by appropriate experts using SAGE techniques. The systematic and practical presentation of the available SAGE technologies in this book, along with examples of applications, should allow both new and experienced readers to glean useful information.
In addition to the growing SAGE databases that are generally available through the internet, there are two attributes of the SAGE data that appear to ensure its future utility. First, the tag data are quantitative and mainly require normalization for total number of sequenced tags, making comparisons between unrelated libraries from different laboratories feasible. Second, future improvements in genome annotation can be used to update and requery old libraries as new questions and insights arise. Some of the earlier barriers to the SAGE technique continue to be minimized by the availability of commercial kits and by the decreasing cost of sequencing the libraries. The book recounts both the modifications and applications of the SAGE technique that reflect the thoughts and concepts behind those who helped to advance it in the genomic era. Although not as widely used as microarrays for transcriptional profiling, the modified SAGE protocols described in this book have greatly expanded its application to include genome annotation and characterization of epigenetic chromatin modifications. At the current pace in this post-genomic era, we envision future editions of this book, including not only modifications and experimental applications, but also practical applications with diagnostic utility for human diseases.