Solid-phase method for differential display of genes expressed in hematopoietic stem cells.
A solid-phase differential display method was designed to analyze differential gene expression in samples with low amounts of mRNA. The principle 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 procedure simplified the purification steps, limited sample loss and enabled 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 characterization of genes expressed in the most immature hematopoietic progenitor cells (CD34+CD38-). The majority of the differentially expressed fragments represented previously uncharacterized sequences.
Røsok
O
O
Norwegian Radium Hospital, Oslo, Norway.
Odeberg
J
J
Rode
M
M
Stokke
T
T
Funderud
S
S
Smeland
E
E
Lundeberg
J
J
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000032:analysis; D001402:B-Lymphocytes / Q000737:chemistry / Q000276:immunology; D001426:Bacterial Proteins; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D018076:DNA, Complementary / Q000138:chemical synthesis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D015870:Gene Expression; D006412:Hematopoietic Stem Cells / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D008562:Membrane Glycoproteins; D008863:Microspheres; D009699:N-Glycosyl Hydrolases / Q000032:analysis; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase; D017422:Sequence Analysis, DNA; D019809:Streptavidin
D015703:Antigens, CD; D000943:Antigens, Differentiation; D001426:Bacterial Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D019809:Streptavidin; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D009699:N-Glycosyl Hydrolases; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D051997:ADP-ribosyl Cyclase 1