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 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.
Nagasawa
Y
Y
Department of Internal Medicine and Therapeutics, Graduate School of Medicine, Osaka University, Osaka, Japan.
Takenaka
M
M
Matsuoka
Y
Y
Imai
E
E
Hori
M
M
D000818:Animals; D004210:Dissection / Q000295:instrumentation* / Q000379:methods; D005615:Freezing; D015971:Gene Expression Regulation, Enzymologic; D005821:Genetic Techniques / Q000295:instrumentation*; D005921:Glomerulonephritis / Q000473:pathology / Q000503:physiopathology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D007678:Kidney Glomerulus / Q000166:cytology / Q000502:physiology*; D007834:Lasers; D008297:Male; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000302:isolation & purification; D051381:Rats; D017207:Rats, Sprague-Dawley; D018800:Thy-1 Antigens / Q000235:genetics; D016212:Transforming Growth Factor beta / Q000235:genetics
D012333:RNA, Messenger; D018800:Thy-1 Antigens; D016212:Transforming Growth Factor beta; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases