Fig. 4.
(A and B) Quantitative analysis of human neuronal nitric-oxide synthase (nNOS) mRNA expression by real-time RT-PCR in the pyloric sphincter of controls and patients with infantile hypertrophic pyloric stenosis (IHPS). Nine forward primers, specific for the alternative first exons 1a–1i of nNOS were used with a common exon 2-specific reverse primer and an internal exon 2-specific 6-carboxy-fluorescein-labeled TaqMan probe. As a parameter for total nNOS mRNA expression, a pair of primers specific for exons 6 and 7, present in all known nNOS cDNAs, were used with an exon 7-specific internal probe. Relative amounts of transcripts were calculated by using standard curves and dividing the expression levels of the different nNOS variants by the expression levels of the neuronal-specific genes Peripherin (A) and microtubule-associated protein 2 (MAP-2) (B) measured in the same RNA preparation. (C) Quantitative analysis of human vasoactive intestinal peptide (VIP) mRNA expression by real-time RT-PCR in the pyloric sphincter of controls and patients with IHPS. Intron-spanning primers for human VIP were used with an internal 6-carboxy-fluorescein-labeled TaqMan probe. Relative amounts of transcripts were calculated by using standard curves and dividing the expression levels of VIP by the expression levels of the neuronal-specific genes PGP9.5, Peripherin, and MAP-2 measured in the same RNA preparation. Results shown are the mean ± SD of pyloric sphincter preparations of 9 controls and 16 IHPS patients. Individual cDNA samples were analyzed in triplicate with a given pair of primers.