ABSTRACT: Headline: application of the oligo-microarray to the analysis of genes modulated by SDF1α chemokine in the human circulating hematopoietic CD34+ progenitors. Summary: 1/ Introduction: The microarray analysis simultaneously explores the expression of thousands messager ribonucleic acids in the cell. The traditional method by reverse transcription requires 50 to 100 micrograms of total initial ribonucleic acids. The human circulating hematopoietic CD34+ progenitors are rare and expressed very few messager ribonucleic acids. SDF1α is a chemokine which binds a specific transmembrane receptor, the CXCR4, expressed on the human circulating hematopoietic CD34+ progenitors. A chemical gradient of SDF1α in the medullary micro-environment is implied in the regulation of the trans-endothelial migration of the hematopoietic CD34+ progenitors who leave bone marrow and then migrate towards peripheral blood. Apart from its role in the cellular mobilization, SDF1α also takes part in survival maintenance, cell cycle promotion and proliferation of hematopoietic progenitors. 2/ objectives: The objective of this work was to apply this technology to the differential analysis of genes modulated by SDF1α chemokine in the human circulating hematopoietic CD34+ progenitors. 3/ Material and methods: With regard to the biological application, CD34+ cells were incubated during sixteen hours in the presence or absence of SDF1α at the concentration of 0,5 ng/ml. For this, four experiments were carried out with CD34+ cells purified from the mononuclear cells obtained from blood of six to eight unmobilized healthy subjects. The total ribonucleic acids of untreated cells from the four experiments were pooled to synthesize a “control” microarray probe. In the same way, the total ribonucleic acids of SDF1α treated cells were used to synthesize a “SDF1α treated” microarray probe. The differential hybridization of an oligo-microarray slide (10 000 genes) was carried out simultaneously with the “control” and “SDF1α treated” probes. The bioinformatics studies and the use of classification algorithms on the results of this microarray analysis made it possible to classify the differential transcripts in intracellular functional groups. 4/ Results an discussion: Our results showed that a double linear amplification with T7 RNA polymerase of complementary retro-transcript desoxyribonucleic acids from total ribonucleic acids of human circulating hematopoietic CD34+ progenitor was necessary to obtain the probe quantity for hybridize an oligo-microarray. Indirect cyanin incorporation of the aminoallyl type during the last stage of in vitro transcription optimized the probe labelling and reduced the experimental skew during the microarray analysis. We showed that under our experimental conditions, SDF1α acted on the expression of genes mainly implied in cell cycle (mitosis induction and proliferation) and cell metabolism (energy and translation). Our microarray results, which were confirmed by quantitative RT-PCR validate the biological ones previously obtained and help us to a better understanding of the intimate regulation mechanism of gene expression modulated by SDF1α. Key words: oligo-microarray, optimization, hematopoietic CD34+ progenitors, chemokine, SDF1α, genomic, transcriptome, cell cycle, quantitative RT-PCR, bioinformatics, principal component analysis, clustering. Keywords: oligo-microarray, optimization, hematopoietic CD34+ progenitors, chemokine, SDF1α, genomic, transcriptome, cell cycle, quantitative RT-PCR, bioinformatics, principal component analysis, clustering.
