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.
Bicknell
G R
GR
University of Leicester Department of Pathology, Leicester Royal Infirmary, U.K.
Shaw
J A
JA
Pringle
J H
JH
Furness
P N
PN
D000199:Actins / Q000378:metabolism; D000818:Animals; D003094:Collagen / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism; D005920:Glomerular Mesangium / Q000378:metabolism; D006801:Humans; D007678:Kidney Glomerulus / Q000166:cytology / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats
D000199:Actins; D012333:RNA, Messenger; D003094:Collagen; D008666:Metalloendopeptidases