title: Rapid expression profiling of brain microvascular endothelial cells by immuno-laser capture microdissection coupled to TaqMan(®) low density array.
abstract: Immuno-laser capture microdissection (immuno-LCM) enables highly selective retrieval of designated cell populations from their in situ locations in complex tissue like the brain. However, the amount of tissue acquired by immuno-LCM is extremely limited, and the RNA purification, amplification and labeling steps necessary for expression analysis by hybridization microarray are tedious and time consuming. This report therefore describes a protocol in which these RNA steps are eliminated altogether, yet allows for global gene profiling. Specifically, immuno-LCM tissue was solubilized and the extract directly subjected to reverse transcription to generate cDNA. Pre-amplification of cDNA was performed next, and then relative expression of 96 different immune-related genes simultaneously determined by quantitative real-time PCR using a microfluidic card TaqMan(®) Low Density Array (TLDA). This protocol was highly reproducible and extremely sensitive, demonstrating high correlation of raw Ct values among both technical and biological replicate samples when using only 1/32 of total pre-amplified cDNA obtained from as little as 500 LCM 'shots.' As this abridged protocol takes only approximately 7h from LCM tissue acquisition to analysis by TLDA, it can prove a very effective tool for both screening and validation purposes when investigating gene regulation in health and disease of the nervous system and other tissues.
authors.affiliation: Blood-Brain Barrier Laboratory, Department of Cell Biology, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030, United States.
authors.forename: Tyler G
authors.initials: TG
authors.lastname: Demarest
authors.forename: Nivetha
authors.initials: N
authors.lastname: Murugesan
authors.forename: Bandana
authors.initials: B
authors.lastname: Shrestha
authors.forename: Joel S
authors.initials: JS
authors.lastname: Pachter
mesh_terms: D000818:Animals; D001921:Brain / Q000098:blood supply / Q000378:metabolism*; D042783:Endothelial Cells / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D061346:Laser Capture Microdissection / Q000379:methods*; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*