REVERSE TRANSCRIPTASE-POLYMERASE CHAIN-REACTION (RT-PCR) - A SENSITIVE METHOD TO EXAMINE BASIC FIBROBLAST GROWTH FACTOR-INDUCED EXPRESSION OF THE EARLY GROWTH-RESPONSE GENE-1 (EGR-1) IN HUMAN UMBILICAL ARTERIAL ENDOTHELIAL-CELLS

Immediate-early genes are expressed upon growth and differentiation in a large variety of cells and species. In the present study we investi gated the effect of basic fibroblast growth factor (bFGF) on early gro wth response gene-1 (egr-1)-mRNA expression in human umbilical arteria l endothelial cells (HUAEC). The detection of this gene in HUAEC was p erformed by Northern blotting and by reverse transcriptase-polymerase chain reaction (RT-PCR). For RT-PCR specific primers for egr-1 and gly ceraldehyde-3-phosphate-dehydrogenase (GAPDH) were constructed and PCR conditions were optimized. bFGF induced a time- and concentration-dep endent increase of egr-1 expression. Maximal expression occurred withi n 30 min of stimulation with bFGF at a concentration of 50-100 ng ml(- 1). RT-PCR gave highly reproducible and specific results. The comparis on of both methods showed comparable results but a higher sensitivity for RT-PCR in detecting the egr-1 mRNA. RT-PCR is an excellent method for detecting the expression of egr-1 mRNA in HUAEC. (C) 1995 Academic Press Limited