SULM – Schweizerische Union für Labormedizin | Union Suisse de Médecine de Laboratoire | Swiss Union of Laboratory Medicine

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S. Mosig1 , P. Büttner1 , M. Hildner1 , H Funke1

1Institute of Vascular Medicine, Dep. of Molecular Hemostaseolgy, Friedrich Schiller University Jena, Bachstr. 18, 07743 Jena, Germany

Microarray analysis has become an important technology in the investigation of disease related changes in gene expression of several tissues. In cases where routine access to tissues primarily involved in pathophysiological processes is not possible gene expression patterns in white blood cells (WBCs) have been used as surrogate markers. For the identification of marker patterns for atherosclerosis WBCs appear particularly suitable as they are in direct contact with cells of the arterial wall. Protocols are needed that allow sampling and shipment under standardized conditions. We tested several commercially available RNA fixation methods which proved unfit. Two methods emerged as useful in which blood is first freed from erythrocytes and WBCs are washed and subsequently lysed in Trizol® within 45 minutes after blood withdrawal. Erythrocyte lysis by hypotonic shock (method 1) showed interindividual differences in the osmotic resistance of WBCs, while isolation of mononuclear cells by Ficoll® centrifugation (method 2) has the disadvantage of contamination by erythroid cells. Using gene expression analysis on Affymetrix HG 133A 2.0 chips, we found several genes related to apoptosis, hypoxia, and inflammation significantly up-regulated in samples treated with erythrocyte lysis buffer compared to samples processed with density gradient centrifugation. This suggests a physiological reaction in the cells in response to this isolation procedure. When we added 1µg/ml Actinomycin D to the Ficoll® supernatant to suppress RNA transcription, we obtained the most reproducible RNA expression patterns. Furthermore, this procedure also yielded good results after overnight storage of unlysed cells at 4°C. Our results demonstrate the feasibility of using gene expression analysis in WBCs for clinical studies.

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