A novel methodology to probe endothelial differential gene expression profile reveals novel genes.
Endothelial dysfunction is a major feature of vascular diseases. A practical, minimally invasive method to effectively "probe" gene transcription for an individual patient's endothelium has potential to "customize" assessment for an individual at risk of vascular disease as well as pathophysiologic insight in an in vivo human, clinical context. Published literature lacks a methodology to identify endothelial differential gene expression in individuals with vascular disease. We describe a methodology to do so. The aim of this study was to specifically utilize (a) cutaneous microvascular biopsy, (b) laser capture microdissection, (c) cDNA amplification, (d) suppression subtractive hybridization, (e) high-throughput sequencing techniques, (f) real-time polymerase chain reaction (PCR), and (g) in combination of these methods, to profile differential gene expression in the context of cardiovascular and cerebrovascular disease. Endothelial cells were obtained by laser capture microdissection from a patient and a healthy sibling's microvascular biopsy tissues. Endothelial RNA was extracted, reverse transcribed, and amplified to ds cDNA. Suppression subtractive hybridization was used to establish an endothelial differential gene expression library. Real-time PCR confirmed SERP1, caspase 8, IGFBP7, S100A4, F85, and F147 up-regulation between 1.4- and 3.47-fold. The authors have successfully established a methodology to profile endothelial differential gene expression and identified six differentially expressed genes. This minimally invasive novel method has potential wide application in the customized assessment of many patients suffering vascular diseases.
Shen
Yi
Y
Department of Neurology, National Neuroscience Institute (SGH Campus), Singapore.
Wilder-Smith
Einar
E
Yu
Esther
E
Ng
Yee Kong
YK
Ling
Eng Ang
EA
Spence
Ian
I
Wong
Meng Cheong
MC
D000328:Adult; D000818:Animals; D001808:Blood Vessels / Q000648:ultrastructure; D002874:Chromosome Mapping; D018076:DNA, Complementary; D030561:Databases, Nucleic Acid; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism* / Q000648:ultrastructure; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D006801:Humans; D007834:Lasers; D008297:Male; D051379:Mice; D016513:Mice, SCID; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary; D012313:RNA; D015252:Deoxyribonucleases, Type II Site-Specific; C040158:GTAC-specific type II deoxyribonucleases