AMPLIFICATION OF SPECIFIC mRNA FROM A SINGLE HUMAN RENAL GLOMERULUS, WITH AN APPROACH TO THE SEPARATION OF EPITHELIAL CELL mRNA

A method has been developed by which single human glomeruli may be plucked from fresh renal biopsies under direct vision, followed by separation of mRNA using oligo-dT-linked paramagnetic beads. The mRNA was amplified by reverse transcription and polymerase chain reaction (RT-PCR). Primers for a variety of human and rat proteins have been developed. The quantity of the amplified cDNA was measured by an enzyme-linked immuno-sorbent assay (ELISA), where biotinylated forward strands of DNA were captured, probed with a fluorescein-conjugated DNA oligomer, and then assayed with an enzyme-linked anti-fluorescein antibody. The cDNA-linked beads are reported to be stable and can be reused with different primer sets, thus forming a "bank' of samples from cases with defined glomerular disorders, which can be used to address new questions as they arise. Using rat glomeruli, a method has been devised which permits at least partial separation of epithelial cell mRNA from mesangial and endothelial cell mRNA.