Effect of tissue processing on the ability to recover nucleic acid from specific renal tissue compartments by laser capture microdissection

The anatomic heterogeneity of the nephron poses obstacles to microdissectio n of individual renal compartments for analysis of gene expression. We have systematically analyzed the effects of fixation time and nuclear staining on the ability to recover nucleic acid from individual renal compartments b y laser capture microdissection (LCM). Formalin-fixed kidney sections from Wistar rats and archival human renal biopsies were used for DNA analysis. F rom 1 to 10 individual glomeruli and from 1 to 10 individual proximal tubul es were captured sequentially onto polymer films. DNA for P-globin could be amplified by PCR from even a single glomerulus or tubule. Optimal conditio ns for DNA amplification were brief (1-or 2-day) formalin fixation. Use of nuclear counterstains, including Weigert's hematoxylin, Harris's hematoxyli n, Mayer's hematoxylin, or methyl green, did nor adversely affect the abili ty to extract and amplify DNA. For RNA extraction, glomeruli and tubules we re microdissected from renal cryostat sections stored for up to 6 months. B y RT-PCR, mRNA expression of the glomerulus-specific gene, Wilms' tumor-1, was identified in as few as 5 microdissected glomeruli and of the tubule-sp ecific gene, aminopeptidase N, in as few as 5 microdissected tubules, with no cross-contamination between renal compartments. Our findings indicate th at the LCM method can successfully microdissect pure glomerular and tubular tissue compartments and that the optimal fixation and staining conditions are those employed routinely for renal biopsies, namely overnight formalin fixation and hematoxylin counterstain for DNA extraction, and cryostat sect ioning with hematoxylin counterstain for RNA extraction. The specificity of LCM coupled with the sensitivity of RT-PCR should prove a powerful tool fo r the analysis of gene expression in specific renal compartments from archi val human renal biopsies. Copyright (C) 2001 S Karger AG, Basel.