Quantification of low abundance natriuretic peptide receptor mRNA in rat tissues

Natriuretic peptides are important regulators of vascular resistance and vo lume and electrolyte homeostasis. The quantification of natriuretic peptide receptor (NPR) mRNA is important for the understanding of the regulation o f this humoral system, but is difficult due to low expression of the NPR mR NA. We report here on the evaluation of a polymerase chain reaction (PCR)-a ided transcript titration assay for quantification of all three NPR subtype s (NPR-A, NPR-B, and NPR-C) mRNA. A multispecific internal standard RNA wit h parts of NPR-A, NPR-B, NPR-C and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nucleotide sequences was constructed and reverse transcription of standard and sample RNA (400 ng) was performed in parallel for all three NP Rs and GAPDH. The specific PCR yielded differently sized products, which we re quantified by high performance liquid chromatography (HPLC). The determi nation of specific mRNA concentrations was not influenced by cDNA input and did not depend on the PCR cycle number. Linearity between sample RNA input and mRNA concentration was demonstrated. Application of the evaluated meth od showed that the NPR-A mRNA expression was the most abundant of the three natriuretic peptide receptor mRNAs in rat lungs, glomeruli and left ventri cles, followed by the NPR-C mRNA and the NPR-B mRNA expression. Thus, the d escribed method allows the reliable quantification of the specific mRNA exp ression of all three NPRs with small amounts of RNA. The presented method m ight foster future research on the regulation of this humoral system in car diovascular and kidney diseases.