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 obse rvation by a laser beam. Laser pressure catapulting (LPC) is a method to pu sh 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 intrave nously into rats. Control and disease model kidneys were obtained. Six-micrometer thick cryostat sections were mo unted onto a 1.35 mu m thin polyethylene membrane. Ten glomeruli were colle cted from 6 mu m frozen sections of rat kidney by LMM and LPC. Isolated glo meruli were used to quantitate the expression of mRNA by real-time polymera se chain reaction (PCR). Results. Transforming growth factor-beta 1 (TGF-beta 1) mRNA was not detect ed 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-beta 1/G3PDH mRNA was 1.89 +/- 0.96 (N = 6). Conclusions. We established methods to isolate glomeruli from standard hist ochemical specimens by LMM and LPC, and to quantify mRNA expression in the targeted glomeruli using real-time PCR. We confirmed the up-regulation of T GF-beta 1 mRNA expression in isolated glomeruli from frozen sections of the anti-Thy1.1 glomerulonephritis model.