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      Effect of Tissue Processing on the Ability to Recover Nucleic Acid from Specific Renal Tissue Compartments by Laser Capture Microdissection

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          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 β-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.

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          Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.

          A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
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            WT-1 is required for early kidney development.

            In humans, germline mutations of the WT-1 tumor suppressor gene are associated with both Wilms' tumors and urogenital malformations. To develop a model system for the molecular analysis of urogenital development, we introduced a mutation into the murine WT-1 tumor suppressor gene by gene targeting in embryonic stem cells. The mutation resulted in embryonic lethality in homozygotes, and examination of mutant embryos revealed a failure of kidney and gonad development. Specifically, at day 11 of gestation, the cells of the metanephric blastema underwent apoptosis, the ureteric bud failed to grow out from the Wolffian duct, and the inductive events that lead to formation of the metanephric kidney did not occur. In addition, the mutation caused abnormal development of the mesothelium, heart, and lungs. Our results establish a crucial role for WT-1 in early urogenital development.
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              HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure.

              Productive infection of human T lymphocytes by HIV-1 is dependent upon proliferation of the infected cell. Nonproliferating quiescent T cells can be infected by HIV-1 and harbor the virus in an inactive state until subsequent mitogenic stimulation. We use a modification of the polymerase chain reaction method, which is both sensitive and quantitative, to demonstrate that HIV-1 DNA synthesis is initiated in infected quiescent T cells at levels comparable with those of activated T cells. However, unlike that of activated T cells, the viral genome is not completely reverse transcribed in quiescent cells. Although this viral DNA structure can persist in quiescent cells as a latent form, it is labile. We discuss the lability of this HIV-1 DNA structure in relation to a "self-restricting persistent infection" by HIV-1 and propose that this may explain the low percentage of infected cells in the circulation of AIDS patients.

                Author and article information

                Nephron Exp Nephrol
                Cardiorenal Medicine
                S. Karger AG
                June 2001
                23 April 2001
                : 9
                : 3
                : 229-234
                aDepartment of Pathology of Columbia University, College of Physicians and Surgeons, and bDepartment of Medicine of Mount Sinai School of Medicine, New York, N.Y., USA
                52616 Exp Nephrol 2001;9:229–234
                © 2001 S. Karger AG, Basel

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                Page count
                Figures: 4, Tables: 1, References: 23, Pages: 6
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/52616
                Technical Report

                Cardiovascular Medicine, Nephrology

                RT-PCR, Laser capture microdissection, Kidney, PCR


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