Measurement of mRNA for E-selectin, VCAM-1 and ICAM-1 by reverse transcription and the polymerase chain reaction.
Stimulation of cultured human umbilical vein endothelial cells by cytokines such as interleukin-1 and tumour necrosis factor induces de novo synthesis and expression of the adhesion molecules E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). In general, alterations in cell surface expression of these molecules are known to be related to increased gene transcription and altered levels of mRNA. The extension of these observations to the study of inflammatory processes in different human organs necessitates the development of techniques for the quantification of mRNA in small tissue samples. Here we present a method for the quantification of mRNA for E-selectin, VCAM-1 and ICAM-1 using reverse transcription and the polymerase chain reaction (RT-PCR). For each molecule of interest a mutant RNA was synthesised consisting of the wild-type sequence deleted of 15-20 bases. The mutant and wild-type RNA sequences are recognised by the same primers, and can therefore be amplified competitively in the same tube by RT-PCR. As the mutant and wild-type RNAs compete for the primers, the amount of wild-type RNA can be determined by the size of the dominant product that results after addition of known quantities of mutant RNA. Using this detection and quantification method we have examined the dose dependency and time course of mRNA accumulation following TNF-alpha stimulation of HUVEC. Similar time-courses of E-selectin, ICAM-1 and VCAM-1 mRNA accumulation were observed by competitive RT-PCR as by laser densitometry of Northern blots. Finally we were able to show that the technique could measure changes in levels of mRNA for these three molecules in human skin biopsies taken at different times during the development of a delayed hypersensitivity response to tuberculin purified protein derivative. This technique should be useful for the study of adhesion molecule mRNA in small tissue culture samples and in biopsies.
Meagher
L
L
Department of Medicine (Rheumatology Unit), Royal Postgraduate Medical School, Hammersmith Hospital, London, UK.
Mahiouz
D
D
Sugars
K
K
Burrows
N
N
Norris
P
P
Yarwood
H
H
Becker-Andre
M
M
Haskard
D O
DO
D001483:Base Sequence; D001706:Biopsy; D015152:Blotting, Northern; D015815:Cell Adhesion Molecules / Q000032:analysis* / Q000235:genetics; D002478:Cells, Cultured; D019040:E-Selectin; D004730:Endothelium, Vascular / Q000737:chemistry; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000032:analysis* / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase; D012867:Skin / Q000737:chemistry; D014471:Umbilical Veins / Q000166:cytology; D019010:Vascular Cell Adhesion Molecule-1
D015815:Cell Adhesion Molecules; D019040:E-Selectin; D012333:RNA, Messenger; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1; D012194:RNA-Directed DNA Polymerase