Semiquantitative RT-PCR analysis of Rac2 and RhoGDI mRNA expression in neutrophils from healthy individuals and SCN patients.

RNA was isolated from neutrophils27 and RT-PCR was performed using specific primers for Rac2 or RhoGDI, respectively. PCR products were separated by agarose gel electrophoresis and blotted onto nylon membranes. Specific products were hybridized with DIG-labeled internal oligonucleotides specific for Rac2 or RhoGDI, respectively, and detection was performed with anti-DIG antibodies and the chemiluminescent substrate CDP-Star. As a control β-actin expression was tested. These results were representative for 5 independent experiments with cells from 2 SCN patients, 2 G-CSF- treated individuals, and 3 untreated healthy individuals. N indicates normal neutrophils; SCN, patients' neutrophils; and d5, in vivo stimulated neutrophils from a healthy donor.