Quantification of low abundance natriuretic peptide receptor mRNA in rat tissues.
Natriuretic peptides are important regulators of vascular resistance and volume and electrolyte homeostasis. The quantification of natriuretic peptide receptor (NPR) mRNA is important for the understanding of the regulation of this humoral system, but is difficult due to low expression of the NPR mRNA. We report here on the evaluation of a polymerase chain reaction (PCR)-aided transcript titration assay for quantification of all three NPR subtypes (NPR-A, NPR-B, and NPR-C) mRNA. A multispecific internal standard RNA with 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 NPRs and GAPDH. The specific PCR yielded differently sized products, which were quantified by high performance liquid chromatography (HPLC). The determination 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 method showed that the NPR-A mRNA expression was the most abundant of the three natriuretic peptide receptor mRNAs in rat lungs, glomeruli and left ventricles, followed by the NPR-C mRNA and the NPR-B mRNA expression. Thus, the described method allows the reliable quantification of the specific mRNA expression of all three NPRs with small amounts of RNA. The presented method might foster future research on the regulation of this humoral system in cardiovascular and kidney diseases.
Höhnel
K
K
Franz Volhard Clinic at the Max Delbrück Centre of Molecular Medicine, University Hospital Charité, Humboldt University, Berlin, Germany.
Dietz
R
R
Willenbrock
R
R
D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D008297:Male; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D017461:Receptors, Atrial Natriuretic Factor / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary; D012333:RNA, Messenger; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D017461:Receptors, Atrial Natriuretic Factor