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

摘要: The anatomic heterogeneity of the nephron poses obstacles to microdissection individual renal compartments for analysis gene expression. We have systematically analyzed effects fixation time and nuclear staining on ability recover nucleic acid from by laser capture (LCM). Formalin-fixed kidney sections Wistar rats archival human biopsies were used DNA analysis. From 1 10 glomeruli proximal tubules captured sequentially onto polymer films. beta-globin could be amplified PCR even a single glomerulus or tubule. Optimal conditions amplification brief (1- 2-day) formalin fixation. Use counterstains, including Weigert's hematoxylin, Harris's Mayer's methyl green, did not adversely affect extract amplify DNA. For RNA extraction, microdissected cryostat stored up 6 months. By RT-PCR, mRNA expression glomerulus-specific gene, Wilms' tumor-1, was identified in as few 5 tubule-specific aminopeptidase N, tubules, with no cross-contamination between compartments. Our findings indicate that LCM method can successfully microdissect pure glomerular tubular tissue optimal are those employed routinely biopsies, namely overnight hematoxylin counterstain sectioning extraction. specificity coupled sensitivity RT-PCR should prove powerful tool specific biopsies.