Vascular gene discovery using laser capture microdissection of human blood vessels and quantitative PCR.
Recent approaches to candidate gene identification and cellular localization have included RNA derived from complex whole tissue profiling on cDNA microarrays followed by in situ hybridization with riboprobes. In this study, the Arcturus PixCell II laser capture microdissection (LCM) system, an argon-based laser-assisted method for the isolation of specific cell types from heterogeneous tissue samples, was used to microdissect the tunica media from normal human arteries and veins (n = 5 in each group). Total RNA was extracted from the sum of 10,000 shots for each blood vessel using the Strataprep MicroKit. RNA was reverse-transcribed, and the resulting cDNA was analyzed using the Applied Biosystems 7700 quantitative PCR system (Q-PCR). Control genes, such as the L-type calcium channel, PECAM (CD-31), and beta-2 microglobulin, were used to assess sample quality and purity. Of 10 laser-captured media samples, five (50%) showed a gene profile that indicated high-quality RNA (abundance of housekeeping genes) and smooth muscle cell enrichment (low levels of PECAM and high levels of the L-type calcium channel). We conclude that the application of the LCM technique to collect smooth muscle cell RNA from the tunica media of human blood vessels can assist in the validation of gene expression and potentially expedite the identification of novel, regulated genes present within vascular smooth muscle.
Stagliano
N E
NE
Millennium Pharmaceuticals Inc, Cambridge, Massachusetts 02139, USA.
Carpino
A J
AJ
Ross
J S
JS
Donovan
M
M
D000199:Actins / Q000235:genetics; D001158:Arteries / Q000166:cytology / Q000502:physiology*; D018076:DNA, Complementary / Q000235:genetics; D004210:Dissection / Q000379:methods; D005821:Genetic Techniques; D006801:Humans; D007834:Lasers; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000235:genetics; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics* / Q000302:isolation & purification; D017540:Tunica Media / Q000502:physiology; D014680:Veins / Q000166:cytology / Q000502:physiology*
D000199:Actins; D018076:DNA, Complementary; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D012313:RNA