Development of Quantitative Reverse Transcription-Polymerase Chain Reaction for the Mesurement of Angiotensin Converting Enzyme mRNA

Huh, Jeong Eun; Kim, Duk Kyung; Choe, Yoon Hyuk; Ryu, Jae Choon; Joo, Shin Bae; Gwon, Hyeon Cheol; Park, Seung Woo; Kim, June Soo; Lee, Sang Hoon; Hong, Kyung Pyo; Park, Jeong Euy; Lee, Won Ro

Abstract

BACKGROUND
The analysis of ACE gene expression in vital to study the role of angiotensin conveting 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 transcription-polymerase chain reaction(RT-PCR) is very effective, sensitive and rapid method of detecting 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 promotor 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, togather with 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 dexamethasone and basis FGF(bFGF). RESULT: The size difference of PCR products from the standard RNA and the extracted target RNA was matched as designed. By using QRT-PCR, there was 1.7*10(8) ACE mRNA molecules in 1 ng of rat lung total RNA. bFGF and dexamethasone upregulated ACE mRNA expression in cultured VSMC.
CONCLUSION
These results suggest that RT-PCR using rcRNA as internal standard is a very useful method for quantitation or semiquantitation of ACE mRNA from a small amount of tissue or cultured cells. Expression of ACE in VSMC can be modulated by various stimuli such as basic FGF and dexamethasone. QRT-PCR could be widely used in the studies of expression of specific human genes.