Tanji N, Ross M D, Cara A, Markowitz G S, Klotman P E, D'Agati V D
Department of Pathology of Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA.
Exp Nephrol. 2001;9(3):229-34. doi: 10.1159/000052616.
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.
肾单位的解剖异质性为剖析单个肾区室以分析基因表达带来了障碍。我们系统分析了固定时间和核染色对通过激光捕获显微切割（LCM）从单个肾区室回收核酸能力的影响。使用来自Wistar大鼠的福尔马林固定肾切片和存档的人类肾活检组织进行DNA分析。将1至10个单个肾小球和1至10个单个近端小管依次捕获到聚合物膜上。即使从单个肾小球或小管中也能通过PCR扩增β-珠蛋白的DNA。DNA扩增的最佳条件是福尔马林短暂（1天或2天）固定。使用包括魏格特苏木精、哈里斯苏木精、迈耶苏木精或甲基绿在内的核复染剂，对提取和扩增DNA的能力没有不利影响。对于RNA提取，从保存长达6个月的肾冷冻切片中显微切割肾小球和小管。通过逆转录聚合酶链反应（RT-PCR），在仅5个显微切割的肾小球中鉴定出肾小球特异性基因威尔姆斯瘤-1的mRNA表达，在仅5个显微切割的小管中鉴定出小管特异性基因氨肽酶N的mRNA表达，且肾区室之间无交叉污染。我们的研究结果表明，LCM方法能够成功地显微切割纯净的肾小球和肾小管组织区室，最佳固定和染色条件是肾活检常规使用的条件，即过夜福尔马林固定和苏木精复染用于DNA提取，冷冻切片和苏木精复染用于RNA提取。LCM的特异性与RT-PCR的敏感性相结合，应成为分析存档人类肾活检组织中特定肾区室基因表达的有力工具。
