Background The analysis of ACE gene expression is vital to study the role of angiotensin converting enzyme ACE in the pathogenesis of cardiovascular disease. Traditionally, levels of individual mRNA expression have been analyzed by semiquantitative Northern blotting, which requires a large quantity of tissue. Therefore, gene expression of a little biopsy specimen from the human heart or atherectomy specimen from the blood vessel cannot be measured easily. Reverse transcriptionpolymerase chain reaction RT-PCR is very effective, sensitive and rapid method of detecting the expression of mRNA, but it is only used in qualitative analysis. Therefore, we established the method of quantitative RT-PCR QRT-PCR using recombinant RNA template as internal standard to measure the expression of ACE.Method Recombinant RNA rcRNA was designed to yield PCR product which differs in size by about 200bp from that of the target RNA. Initially, spacer gene, which was composed of ACE sense primer, antisense primer, T7 promoter and poly dT tail with glutathione transferase GSTM gene of 180bp in the middle, was constructed. Then, standard rcRNA was obtained by in vitro transcription. Target RNA was mixed with rcRNA and amplified by PCR, together with 32 P-dCTP. PCR products were analyzed by gel electrophoresis. For quantitation, either gel was cut and radioactivity was counted or gel was dried and exposed to X-ray film and density was measured using image densitometer. We carried out semiquantitative RT-PCR to study the modulation of ACE expression in vascular smooth muscle cell VSMC by dexame-thasone and basic FGF bFGF .
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