Quantitation of inflammatory und proliferative genes as disease markers in laser-microdissected, formalin-fixed and paraffinized glomeruli from human renal biopsies

Organ biopsies from kidney, liver, intestine or heart are presently only evaluated for pathologic lesions using formalin-fixation and paraffin-embedding. Changes in pattern and levels of gene expression underlying and preceding these lesions have been disregarded, since a quantitative assay system using minute paraffinized tissue samples with minimal amounts of RNA is currently unavailable. This paper describes such an assay using formalin-fixed, paraffinized biopsies from human glomeruli after laser-microdissection. Choosing proliferative glomerular lesions expected to express platelet-derived growth factor-β receptor (PDGF-βR), we investigated whether vascular cell adhesion molecule-1 (VCAM-1), a marker of the inflammatory signal pathway, was coexpressed, indicating the potential to progress an already existing lesion. RNA extraction procedures including DNAse pretreatment were pretested on microdissected mouse glomeruli. β-actin, not upregulated in cell culture by TNF (tumor necrosis factor) or PDGF stimulation, served as housekeeping gene. By qualitative real-time (RT)-PCR, transcription of both genes was detectable using only one microdissected glomerular section. Quantitative PCR for VCAM-1 revealed unsuspected increased levels in the same cases positive by qualitative RT-PCR, not detectable by morphologic analysis or immunohistochemistry. Thus, quantitative gene expression analysis is possible in paraffinized biopsies from minute tissue samples and will provide such important information as unexpected gene expression causing a potential progression of an existing lesions.