Analyses Of Global Gene Expression In Endothelial Cells Derived From Lymphoma

Part I Establishment of the empirical method[Objective]Combined application of LCM and cDNA microarray for analyzing global gene expression profiles in endothelial cells derived from lymphoma.[Methods]we select a tissue fixation and isolation of RNA from laser-captured cells retrieved from frozen sections previously subjected to immunohistochemistry and subsequently subjected to linear amplification and cDNA array analysis.[Results]Our results demonstrated that zinc-fixative provided the best protection for RNA integrity under experimental conditions mimicking LCM handling, Anti-CD34 antibody strongly stained both small and large vessels of lymph node with minimal staining of surrounding tissues and nanograms amounts of total RNA isolated from about 3000 VEC can be amplified linearly and that the amplified RNA could generate enough signal intensity for GeneChip analysis.[Conclusion]Our data suggested the current method provided a powerful tool to compare gene expression patterns of endothelial cells derived from 1ymphoma. Part II Validation of experiment[Objective]Validate the experiment of Part I[Methods]Semiquantitative RT-PCR was performed to evaluate the purity of LCM-isolated VEC. We used pair-wise scatter-plots to ensure stability of the amplification of RNA from zinc-fixed tissues, 1μl of amplified RNA and unamplified RNA were used as template for semi-quantitative PCR to evaluate the amplification bias and In situ hybridization was performed to validate the Microarray Results.[Results]Transcripts of the B-lymphocyte antigen CD19 was undetectable in purified VECs while CD34 showed abundant amplification. cDNA amplification did not alter the relative abundance of three transcripts, which suggested the cDNA amplification did not lead bias. The correlation coefficient was 0.96 according to the analyzing of pair-wise scatter-plots. The expression of HERV in lymphoma endothelium was confirmed by In situ hybridization.[Conclusion]The combination of the LCM and cDNA microarray techniques are effective used for our research work.