Development and characterization of a novel method to analyze global gene expression profiles in endothelial cells derived from primary tissues.
Homogenous cells separated directly from clinical samples with laser capture microdissection (LCM) contain their original transcript messages, which accurately reflects the physiological and pathological changes of the cell before fixation. Analysis of such samples provides a direct understanding of in vivo physiological and pathological processes. The development of modern large-scale analytical techniques have given us a chance to determine the entire transcript profile of interesting cells. Therefore, LCM and gene expression microarray analysis are becoming common tools across a broad range of disciplines, particularly in the basic and clinical biomedical sciences. However, the combination of these technologies is limited in the study of vascular endothelial cell (VEC) since the precise location and separation of VECs is not easily realized and the requirement for relatively large amount of intact RNA for labeling and hybridization is not easily available. In this report, we describe an approach for tissue fixation and isolation of RNA from laser-captured cells retrieved from frozen sections, which had previously been subjected to immunohistochemistry and subsequently subjected to linear amplification and gene expression microarray analysis. Our results demonstrate that nanogram quantities of total RNA isolated from about 3,000 VECs can be amplified and that the amplified RNA can generate enough signal intensity for GeneChip analysis. analysis.
Bai
Xiangyang
X
Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China.
Huang
Mei
M
Wu
Jingyi
J
Huang
Xiaoyuan
X
Yan
Lingling
L
Lu
Yunping
Y
Wang
Shixuan
S
Xu
Gang
G
Zhou
Jianfeng
J
Ma
Ding
D
D018076:DNA, Complementary / Q000235:genetics; D042783:Endothelial Cells / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D012333:RNA, Messenger / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D018076:DNA, Complementary; D012333:RNA, Messenger