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 microdissection 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 by laser capture microdissection (LCM). Formalin-fixed kidney sections from Wistar rats and archival human renal biopsies were used for DNA analysis. From 1 to 10 individual glomeruli and from 1 to 10 individual proximal tubules were captured sequentially onto polymer films. DNA for beta-globin could be amplified by PCR from even a single glomerulus or tubule. Optimal conditions for DNA amplification were brief (1- or 2-day) formalin fixation. Use of nuclear counterstains, including Weigert's hematoxylin, Harris's hematoxylin, Mayer's hematoxylin, or methyl green, did not adversely affect the ability to extract and amplify DNA. For RNA extraction, glomeruli and tubules were microdissected from renal cryostat sections stored for up to 6 months. By 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-specific gene, aminopeptidase N, in as few as 5 microdissected tubules, with no cross-contamination between renal compartments. Our findings indicate that 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 sectioning with hematoxylin counterstain for RNA extraction. The specificity of LCM coupled with the sensitivity of RT-PCR should prove a powerful tool for the analysis of gene expression in specific renal compartments from archival human renal biopsies.
Tanji
N
N
Department of Pathology of Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA.
Ross
M D
MD
Cara
A
A
Markowitz
G S
GS
Klotman
P E
PE
D'Agati
V D
VD
D000818:Animals; D015870:Gene Expression; D006801:Humans; D007668:Kidney / Q000737:chemistry*; D007678:Kidney Glomerulus / Q000737:chemistry; D007687:Kidney Tubules, Proximal / Q000737:chemistry; D009696:Nucleic Acids / Q000032:analysis*; D016133:Polymerase Chain Reaction; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013194:Staining and Labeling / Q000379:methods*; D013997:Time Factors; D016707:Tissue Fixation / Q000379:methods*
D009696:Nucleic Acids