Microtranscriptomics and microproteomics of human glomerular diseas

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This project was aimed to establish platform for transcriptomics and proteomics of human kidney biopsy samples from kidney disease patients.1) Human glomerulus cDNA library was prepared and approximately 6,000 clones were sequenced. The normal human glomerular transcriptome was analyzed by DNA microarray technique and the database was created by annotating the glomerular genes. By the analysis several genes were demonstrated as those uniquely expressed in the glomerulus. Their expression was localized by in situ hybridization and immunohistochemistry techniques.2) The proteome of normal human glomerulus was analyzed by 2D gel electrophoresis and nanoLC-ESI TOF MS. More than 7000 proteins originated from about 3000 genes were identified in the glomerulus and a glomerular proteome database was created in association with immunohistochemistry image data provided from the Human Protein Atlas project of Human proteome organization (HUPO).3) To establish procedures for microtranscriptomics and microproteomics to analyze glomerulus in human kidney biopsy samples, glomerular sections were microdissected and mRNA and protein were isolated. From 50 glomerular sections, about one pg each of RNA and protein was isolated and was applicable for microanalysis. Contamination of human keratin and co-existence of human plasma proteins in the glomerular protein samples were also established.