Isolating vessels from the mouse brain for gene expression analysis using laser capture microdissection.
Studies of gene expression often examine a pool of RNA extracted from the diverse cell types making up a tissue. We have developed a method for isolating vessels from the brain in order to understand the changes occurring in the vessels during the pathogenesis of cerebral malaria. Vessels were visualised by incubating sections of mouse brain with a substrate for alkaline phosphatase. Vessels were collected by laser capture microdissection and the specificity was monitored by measuring the expression of cell-specific markers. RNA from the captured vessels was highly enriched in mRNA for genes associated with endothelial cells and pericytes. Measurement of indoleamine 2,3-dioxygenase mRNA indicated it was possible to detect changes in gene expression, due to malaria infection, occurring specifically within the vessels. Laser capture microdissection can be used to study changes in gene expression occurring at the blood-brain barrier.
Department of Pathology, Blackburn Bldg. D06, University of Sydney, Camperdown N.S.W. 2006, Australia. helenb@med.usyd.edu.au
Helen J
HJ
Ball
Brent
B
McParland
Catherine
C
Driussi
Nicholas H
NH
Hunt
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D001808:Blood Vessels / Q000378:metabolism* / Q000469:parasitology / Q000503:physiopathology; D001812:Blood-Brain Barrier / Q000235:genetics / Q000276:immunology; D001921:Brain / Q000098:blood supply* / Q000469:parasitology / Q000503:physiopathology; D002135:Calcium-Binding Proteins / Q000235:genetics; D004396:Coloring Agents; D004195:Disease Models, Animal; D004210:Dissection / Q000379:methods*; D004730:Endothelium, Vascular / Q000378:metabolism / Q000469:parasitology / Q000503:physiopathology; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology*; D006651:Histocytochemistry / Q000379:methods; D007834:Lasers; D016779:Malaria, Cerebral / Q000235:genetics* / Q000473:pathology / Q000503:physiopathology; D051379:Mice; D008808:Mice, Inbred CBA; D009414:Nerve Growth Factors / Q000235:genetics; D020286:Pericytes / Q000378:metabolism / Q000469:parasitology / Q000473:pathology; D012333:RNA, Messenger / Q000302:isolation & purification*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; D014366:Tryptophan Oxygenase / Q000235:genetics
D015415:Biomarkers; D002135:Calcium-Binding Proteins; D004396:Coloring Agents; D009414:Nerve Growth Factors; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; D014366:Tryptophan Oxygenase; D000469:Alkaline Phosphatase