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-polymerase 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 reverse transcribed and amplified with dilutions of a competitor RNA fragment used as a control. We constructed a single synthetic RNA competitor for ANF, 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 collected a right atrial sample from each patient before the start of cardiopulmonary 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 radioactive procedure for the quantification of natriuretic peptide gene expression and is particularly suitable for evaluation of gene expression in small myocardial samples. Our data also suggest that: 1) there is a significant relationship 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.
Iascone
M R
MR
CNR Istituto di Fisiologia Clinica, Pisa, Italy.
Vittorini
S
S
Collavoli
A
A
Cupelli
A
A
Kraft
G
G
Biagini
A
A
Clerico
A
A
D000199:Actins / Q000235:genetics; D009320:Atrial Natriuretic Factor / Q000235:genetics*; D001667:Binding, Competitive; D015152:Blotting, Northern; D002675:Child, Preschool; D005260:Female; D015870:Gene Expression; D006325:Heart Atria / Q000737:chemistry; D006330:Heart Defects, Congenital / Q000378:metabolism* / Q000601:surgery; D006801:Humans; D007223:Infant; D008297:Male; D009206:Myocardium / Q000737:chemistry; D020097:Natriuretic Peptide, Brain / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D000199:Actins; D012333:RNA, Messenger; D020097:Natriuretic Peptide, Brain; D009320:Atrial Natriuretic Factor