Rapid expression profiling of brain microvascular endothelial cells by immuno-laser capture microdissection coupled to TaqMan庐 Low Density Array
- 作者：Tyler G. Demarest ; Nivetha Murugesan ; Bandana Shrestha ; Joel S. Pachter ; pachter@nso1.uchc.edu
- 关键词：Laser capture microdissection ; Brain microvascular endothelial cells ; Gene expression profiling ; Microarray ; TLDA
- 刊名：Journal of Neuroscience Methods
- 出版年：2012
- 期刊代码：33_01650270
- 类别：neu
- 出版时间：15 May, 2012
- 卷：206
- 期：2
- 页码：200-204
- 文件大小：284 K
摘要
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庐 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 鈥榮hots.鈥?As this abridged protocol takes only approximately 7 h 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.