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.
