SOLID-PHASE METHOD FOR DIFFERENTIAL DISPLAY OF GENES EXPRESSED IN HEMATOPOIETIC STEM-CELLS

A solid-phase differential display method was designed to analyze diff erential gene expression in samples with low amounts of mRNA. The prin ciple was based on using a biotinylated probe to capture the mRNA and priming both the first-strand synthesis and the subsequent polymerase chain reaction step. Coupling the mRNA to a solid phase during the pro cedure simplified the purification steps, limited sample loss and enab led rapid handling of mRNA. DNA contamination was also minimized when the mRNA was bound to a solid phase. Optimization of the differential display method was achieved by analyzing both the enzymatic conditions and the required cell amounts. The approach was used for the characte rization of genes expressed in the most immature hematopoietic progeni tor cells (CD34(+)CD38(-)). The majority of the differentially express ed fragments represented previously uncharacterized sequences.