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

Mol Cell Probes. 1995 Aug;9(4):215-22. doi: 10.1016/s0890-8508(95)90070-5.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Cells, Cultured
  • DNA Primers
  • DNA-Binding Proteins / biosynthesis*
  • DNA-Binding Proteins / genetics
  • Early Growth Response Protein 1
  • Endothelium, Vascular / metabolism*
  • Fibroblast Growth Factor 2 / pharmacology*
  • Gene Expression
  • Humans
  • Immediate-Early Proteins*
  • Molecular Sequence Data
  • Polymerase Chain Reaction / methods
  • RNA, Messenger / analysis
  • Transcription Factors / biosynthesis*
  • Transcription Factors / genetics

Substances

  • DNA Primers
  • DNA-Binding Proteins
  • EGR1 protein, human
  • Early Growth Response Protein 1
  • Immediate-Early Proteins
  • RNA, Messenger
  • Transcription Factors
  • Fibroblast Growth Factor 2