Establishment of a quantitative RT-pCR for detection of vascular cell adhesion molecule-1 transcripts in endothelial cells after stimulation with advanced glycation endproducts.
Advanced glycation endproducts (AGE) are supposed to increase endothelial expression of adhesion molecules like vascular cell adhesion molecule-1 (VCAM-1) by inducing an intracellular stress with subsequent activation of nuclear transcription factor NF-kappa-B. Quantitative analysis of VCAM-1-transcription has not been demonstrated concerning this topic. Thus, the aim of this study was to establish quantitative reverse transcription polymerase chain reaction (RT-PCR) assays using a spacer gene in order to measure the amounts of specific mRNA for VCAM-1 in human umbilical vein endothelial cells (HUVEC) which were stimulated with AGE-albumin (AGE-BSA). A recombinant RNA-standard was synthesized and used as internal RT-PCR standard. The amount of VCAM-1-mRNA in unstimulated HUVEC was found to be 2.2 +/- 2.7 copies per cell. After stimulation with AGE-BSA, mRNA-levels were elevated to 38.9 +/- 10.9 copies per cell. Positive controls (stimulated with lipopolysaccharide) revealed mRNA-levels of 78.7 +/- 27.5 copies per cell. We conclude that quantitative RT-PCR using the spacer gene technique is a valid and reliable method for the measurement of small amounts of specific
Kunt
T
T
Department for Internal Medicine and Endocrinology, University of Mainz, Langenbeckstr. 1, D-55131 Mainz, Germany.
Forst
T
T
Wilhelm
A
A
Zschäbitz
A
A
Göpfert
A
A
Kann
P
P
Pfuetzner
A
A
Harzer
O
O
Engelbach
M
M
Beyer
J
J
Stofft
E
E
D002478:Cells, Cultured; D042783:Endothelial Cells / Q000737:chemistry; D004730:Endothelium, Vascular / Q000166:cytology; D017127:Glycation End Products, Advanced / Q000737:chemistry*; D006801:Humans; D012333:RNA, Messenger / Q000032:analysis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D019010:Vascular Cell Adhesion Molecule-1 / Q000032:analysis* / Q000235:genetics
D017127:Glycation End Products, Advanced; D012333:RNA, Messenger; D019010:Vascular Cell Adhesion Molecule-1; C081628:advanced glycation end products-bovine serum albumin; D012710:Serum Albumin, Bovine