Quantification of constitutive endothelial and inducible nitric oxide synthase mRNA by competitive reverse transcription-polymerase chain reaction

Abstract

Nitric oxide (NO) participates in the general homeostatic control of the vasculature, and it is involved in the process of vascular remodelling. NO in particular inhibits the proliferation of vascular smooth muscle cells and has been shown to possess antiatherogenic properties. Two important molecules control NO synthesis, namely constitutive endothelial (ecNOS) and inducible (iNOS) nitric oxide synthase. To investigate the regulation of the ecNOS and iNOS mRNA expression in various tissues, we describe the design and validation of a reliable and efficient competitive RT-PCR approach for quantification of ecNOS and iNOS mRNA in rat tissue. Prior to reverse transcription, the total RNA was supplemented with internal standard RNA-competitors, which were constructed by a modified site-directed mutagenesis followed by in vitro transcription using T7-polymerase. This technique allows the easy and fast (within a single day) construction of an internal, recombinant RNA-fragment without the use of cloning techniques. Only two additional “linker” primers containing the sequence of T7-promoter, the primers used for the wild type of ecNOS and iNOS mRNA and the primers of a spacer gene are needed. In addition, all steps of the procedure can be streamlined by convenient commercially available kits. We conclude that the described technique is a valid and reliable method for the absolute quantification of small amounts of specific mRNA.