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.

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