Quantitative polymerase chain reaction (PCR) to analyze messenger-RNA expression in tissue obtained by directional atherectomy.
There is a widely recognized need to evaluate gene expression in human vascular plaque tissue. Directional atherectomy made it possible to sample plaque tissue from primary stenoses and subsequently from restenoses from one individual. Conventional approaches to analyze mRNA content of lesions, such as Northern blot analysis, and slot blot analysis are not sensitive enough to evaluate mRNA levels in atherectomy specimens limited by low cell number or low copy number per cell. The purpose of this study was to investigate whether gene expression, as reflected in mRNA copy number, in atherectomy biopsies, could be sufficiently analyzed by quantitative polymerase chain reaction (PCR). The authors applied PCR to detect platelet-derived growth factor-A mRNA expression in 12 human lesions sampled percutaneously by directional atherectomy. After reverse transcription, specific amplification of the resulting cDNA was performed. This was successful with cDNA from less than 0.5 microgram of total cellular RNA. To quantitate mRNA content of specimens, the authors coamplified cDNA copies of mRNA from lesions and from a synthetic reference RNA in the same reaction vessel. Quantitative PCR is best applied if tissue is more than 45 mg in weight and of high cellularity with low calcification. This method allows quantitation of mRNA in human primary and restenotic lesions, and it complements histochemical approaches and in situ hybridization of coronary and peripheral atherectomy specimens.
Department of Internal Medicine, University of Munich, Germany.
R
R
Köster
U
U
Windstetter
J M
JM
Gulizia
P
P
Uberfuhr
G
G
Baumann
B
B
Höfling
D001011:Aorta / Q000473:pathology; D001161:Arteriosclerosis / Q000235:genetics* / Q000473:pathology; D017073:Atherectomy / Q000379:methods; D017225:Atherectomy, Coronary; D001483:Base Sequence; D001706:Biopsy; D015139:Blotting, Southern; D003331:Coronary Vessels / Q000473:pathology* / Q000601:surgery; D005263:Femoral Artery / Q000473:pathology* / Q000601:surgery; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D010982:Platelet-Derived Growth Factor / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis*
D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; C075278:platelet-derived growth factor A