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- Title
Quantitation of mRNA expression in glomeruli using laser-manipulated microdissection and laser pressure catapulting.
- Authors
Nagasawa, Yasuyuki; Takenaka, Masaru; Matsuoka, Yasuko; Imai, Enyu; Hori, Masatsugu
- Abstract
Quantitation of mRNA expression in glomeruli using laser-manipulated microdissection and laser pressure catapulting. Background. Laser-manipulated microdissection (LMM) is a method to cut out a single cell or limited tiny region from a specimen under microscopic observation by a laser beam. Laser pressure catapulting (LPC) is a method to push up and collect samples that were microdissected using a strong laser. Methods. To induce experimental glomerulonephritis, anti-Thy1.1 monoclonal antibody (OX-7) was injected intravenously into rats. Control and disease model kidneys were obtained. Six-micrometer thick cryostat sections were mounted onto a 1.35 μm thin polyethylene membrane. Ten glomeruli were collected from 6 μm frozen sections of rat kidney by LMM and LPC. Isolated glomeruli were used to quantitate the expression of mRNA by real-time polymerase chain reaction (PCR). Results. Transforming growth factor-β1 (TGF-β1) mRNA was not detected in glomeruli isolated by the LMM and the LPC methods on day 0, although G3PDH mRNA was measurable in the same samples. On day 7 after the treatment with OX-7, the ratio of TGF-β1/G3PDH mRNA was 1.89 ± 0.96 (N = 6). Conclusions. We established methods to isolate glomeruli from standard histochemical specimens by LMM and LPC, and to quantify mRNA expression in the targeted glomeruli using real-time PCR. We confirmed the up-regulation of TGF-β1 mRNA expression in isolated glomeruli from frozen sections of the anti-Thy1.1 glomerulonephritis model.
- Subjects
MESSENGER RNA; KIDNEY glomerulus; MICRODISSECTION; CYTOKINES
- Publication
Kidney International, 2000, Vol 57, Issue 2, p717
- ISSN
0085-2538
- Publication type
Article
- DOI
10.1046/j.1523-1755.2000.00894.x