Open AccessSolid-Phase Method for Differential Display of Genes Expressed in Hematopoietic Stem Cells
Author(s) -
Øystein Røsok,
Jacob Odeberg,
Miriam Rode,
Trond Stokke,
Steinar Funderud,
Erlend B. Smeland,
Joakim Lundeberg
Publication year - 1996
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/96211rr02
Subject(s) - haematopoiesis , stem cell , gene , biology , differential display , microbiology and biotechnology , genetics , gene expression
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.