Quantitation of mRNA expression in glomeruli using laser-manipulated microdissection and laser pressure catapulting

Kidney Int. 2000 Feb;57(2):717-23. doi: 10.1046/j.1523-1755.2000.00894.x.


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 microm thin polyethylene membrane. Ten glomeruli were collected from 6 microm 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-beta1 (TGF-beta1) 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-beta1/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-beta1 mRNA expression in isolated glomeruli from frozen sections of the anti-Thy1.1 glomerulonephritis model.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Dissection / instrumentation*
  • Dissection / methods
  • Freezing
  • Gene Expression Regulation, Enzymologic
  • Genetic Techniques / instrumentation*
  • Glomerulonephritis / pathology
  • Glomerulonephritis / physiopathology
  • Glyceraldehyde-3-Phosphate Dehydrogenases / genetics
  • Kidney Glomerulus / cytology
  • Kidney Glomerulus / physiology*
  • Lasers
  • Male
  • Paraffin Embedding
  • Polymerase Chain Reaction
  • RNA, Messenger / analysis*
  • RNA, Messenger / isolation & purification
  • Rats
  • Rats, Sprague-Dawley
  • Thy-1 Antigens / genetics
  • Transforming Growth Factor beta / genetics


  • RNA, Messenger
  • Thy-1 Antigens
  • Transforming Growth Factor beta
  • Glyceraldehyde-3-Phosphate Dehydrogenases