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 investigated the effect of basic fibroblast growth factor (bFGF) on early growth response gene-1 (egr-1)-mRNA expression in human umbilical arterial endothelial cells (HUAEC). The detection of this gene in HUAEC was performed by Northern blotting and by reverse transcriptase-polymerase chain reaction (RT-PCR). For RT-PCR specific primers for egr-1 and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) were constructed and PCR conditions were optimized. bFGF induced a time- and concentration-dependent increase of egr-1 expression. Maximal expression occurred within 30 min of stimulation with bFGF at a concentration of 50-100 ng ml-1. RT-PCR gave highly reproducible and specific results. The comparison 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.
D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D051766:Early Growth Response Protein 1; D004730:Endothelium, Vascular / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D015870:Gene Expression; D006801:Humans; D017874:Immediate-Early Proteins; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics
D017931:DNA Primers; D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D017874:Immediate-Early Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D016222:Fibroblast Growth Factor 2