Generation of Kidney Transcriptomes Using Serial Analysis of Gene Expression

Abstract

Chronic renal disease initiation and progression remain incompletely understood. Genomewide expression monitoring should clarify the mechanisms which cause progressive renal disease by determining how clusters of genes coordinately change their activity. Serial analysis of gene expression (SAGE) is a technique of expression profiling which permits simultaneous and quantitative analysis of 9- to 13-bp sequence tags that correspond to unique mRNAs. Key principles of the technique are use of PCR in a manner to minimize distortion and serial concatenation of tags which facilitates sequencing and permits identification of many expressed genes in a single cDNA molecule. Tags are extracted from many concatenated sequences, counted using software, and identified by comparison with existing gene databases. In aggregate, gene expression profiles generated from a tag library comprise a transcriptome which represents a comprehensive and quantitative profile of genes expressed at the time of analysis. These global snapshots of gene expression patterns can better define basic cell biology and provide insights into disease pathogenesis by simultaneously determining the net consequences of gene-gene and gene-environment interactions on expression of thousands of genes. Rather than applying a priori assumptions (i.e., hypothesis testing), transcriptome analysis is hypothesis generating and requires no prior knowledge of gene expression. SAGE kidney transcriptomes, from normal animals and animals with progressive kidney disease, are being produced and can be analyzed for novel pathogenetic mechanisms. The use of SAGE and other genomic and proteomic tools should result in a better understanding of kidney disease pathogenesis and in identification of new therapeutic targets.

Serial analysis of gene expression in mouse kidney following angiotensin II administration

“…Although several statistical methods are now available for the quantitative comparison of SAGE libraries (3,10,27,32,35,36,60,74), with the various tests differing to some degree in their specificity and sensitivity (33,48), the optimal approach still remains controversial (50). Moreover, since each SAGE library provides only one measurement, the biological interanimal variation and the accuracy of experimentation cannot be assessed, which necessitates subsequent verification of results by an independent method.…”

Discovery of Genes Related to Diabetic Nephropathy in Various Animal Models by Current Techniques

“…The method is based on the isolation of unique sequence tags (9- to 13-bp in length) from individual mRNAs and concatenation of tags serially into long DNA molecules for lump-sum sequencing [58]. So far, the usage of SAGE has been restricted to the expression profiling of kidney transcriptomes [59], in particular the renal collecting duct cells of mice subjected to potassium depletion [60], and comparative gene analyses between various cell types, such as glomerular versus aortic endothelia [61]. In this regard, the advent of high-throughput next generation sequencers, such as the 1G Genome Analyzer (Illumina/Saleza) and SOLID System (Applied Biosystems) have greatly facilitated gene expression profiling by SAGE tag with higher efficiency and lower costs.…”

Windows on Renal Biopsy Interpretation

“…While the emphasis has been on those genes that are increased, some genes that may be protective (such as MMPs) may be decreased. Therefore, microarray and Sage techniques coupled with detailed genetic studies will likely represent necessary and informative steps in defining those genes that are activated or are inhibited in disease categories or in the individual renal disease patient (21)(22)(23).…”