A rapid procedure for the quantitation of natriuretic peptide RNAs by competitive RT-PCR in congenital heart defects

We report an original application of quantitative reverse transcription-pol ymerase chain reaction (Q.RT-PCR) for the evaluation of atrial natriuretic factor (ANF), brain natriuretic peptide (BNP) and beta-actin mRNA in small atrial samples (10-15 mg). A fixed amount of total RNA was simultaneously r everse transcribed and amplified with dilutions of a competitor RNA fragmen t used as a control. We constructed a single synthetic RNA competitor for A NF, BNP and beta-actin. The competitors and targets shared the same primer sequences but yielded PCR products of different sizes. We have investigated ANF, BNP and beta-actin gene expression in patients affected by congenital heart defects (CHD) during the surgical correction of the defect. We colle cted a right atrial sample from each patient before the start of cardiopulm onary bypass. We studied 14 patients affected by tetralogy of fallot (no. 6 ), complex CHD (no. 4), and ventricular septal defect (no. 4). The results indicate that the Q.RT-PCR represents a simple, highly specific, non radioa ctive procedure for the quantification of natriuretic peptide gene expressi on and is particularly suitable for evaluation of gene expression in small myocardial samples. Our data also suggest that: 1) there is a significant r elationship between the levels of ANF, BNP and beta-actin mRNA and the type of CHD; 2) the levels of BNP mRNA are more variable than ANF; 3) the beta- actin seems to be unsuitable for normalising cardiac gene expression in CHD because mRNA basal levels of this protein vary greatly among patients with different type of CHD. (J. Endocrinol. Invest. 22: 835-842, 1999) (C) 1999 , Editrice Kurtis.