Serial microanalysis of renal transcriptomes

Abstract

Large-scale gene expression studies can now be routinely performed on macroamounts of cells, but it is unclear to which extent current methods are valuable for analyzing complex tissues. In the present study, we used the method of serial analysis of gene expression (SAGE) for quantitative mRNA profiling in the mouse kidney. We first performed SAGE at the whole-kidney level by sequencing 12,000 mRNA tags. Most abundant tags corresponded to transcripts widely distributed or enriched in the predominant kidney epithelial cells (proximal tubular cells), whereas transcripts specific for minor cell types were barely evidenced. To better explore such cells, we set up a SAGE adaptation for downsized extracts, enabling a 1,000-fold reduction of the amount of starting material. The potential of this approach was evaluated by studying gene expression in microdissected kidney tubules (50,000 cells). Specific gene expression profiles were obtained, and known markers (e.g., uromodulin in the thick ascending limb of Henle's loop and aquaporin-2 in the collecting duct) were found appropriately enriched. In addition, several enriched tags had no databank match, suggesting that they correspond to unknown or poorly characterized transcripts with specific tissue distribution. It is concluded that SAGE adaptation for downsized extracts makes possible large-scale quantitative gene expression measurements in small biological samples and will help to study the tissue expression and function of genes not evidenced with other high-throughput methods.

A large-scale screening of the normalized mammalian mitochondrial gene expression profiles

“…Taken together, over 17 . 7 million SAGE tags derived from SAGE libraries generated with commonly used NlaIII (Velculescu et al, 1995) and Sau3A I (Virlon et al, 1999) anchoring enzymes were subjected to the analysis. Clustering analysis has allowed identification of some common patterns in the profiles of mitochondrial gene expression and the potential biological significance of variations in the total mitochondrial gene expression is discussed.…”

Transcriptome of a mouse kidney cortical collecting duct cell line: Effects of aldosterone and vasopressin

“…To achieve these goals, we used the only powerful tool presently available, i.e., serial analysis of gene expression (SAGE), which allows a quantitative monitoring of all mRNA transcripts or transcriptome, of a cell (21). The transcriptome of the collecting duct, which is made of at least two cell types (principal and intercalated cells), has been recently reported (22). For methodological reasons (limitation in cell isolation and in the amount of mRNA that can be obtained), it is not yet feasible to analyze the transcriptome of the principal cells per se, directly isolated from a CCD.…”

Gene expression profiles in normal and Otx2 −/− early gastrulating mouse embryos

“…Thirty embryos were used in each case. Libraries were generated by using the SAGE adaptation for downsized extracts (SADE) method (7). Poly(A) RNAs were isolated through hybridization to oligo(dT) 25 covalently bound to magnetic beads by using Dynabeads mRNA direct kit (Dynal, Great Neck, NY).…”

“…We therefore used the serial analysis of gene expression (SAGE) method, which provides quantitative gene expression profiles (6), and was recently scaled-down to make possible the analysis of microdissected structures (7). Two SAGE libraries were constructed, from wild-type (WT) and Otx2 Ϫ/Ϫ embryos at early gastrulation (6.5 dpc), the stage at which Otx2 function is primarily required.…”