Fig. 1.

Quantitative analysis of human nNOS mRNA expression by real-time RT-PCR in the pyloric sphincter of controls and patients with IHPS. A set of nine forward primers, specific for the nine alternative first exons of nNOS (exon 1a–1i), 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 globally expressed GAPDH housekeeping gene (A) or the neuronal-specific gene PGP9.5 (B) 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. (C) Relative amounts of the different alternative first exons of nNOS normalized against GAPDH in controls and patients with IHPS as determined by quantitative RT-PCR.