Localization of inducible nitric oxide synthase mRNA in inflamed gastrointestinal mucosa by in situ reverse transcriptase-polymerase chain reaction

Abstract

Immunohistochemistry has been critical in determining the tissue localization of inducible nitric oxide synthase (iNOS). However, this technique suffers from nonspecific staining which may lead to false-positive results and the failure of antisera to recognize iNOS from different species. We developed a technique to determine the localization of iNOS mRNA, as opposed to protein, in tissue sections using an in situ RT-PCR (IS RT-PCR) technique. Sections of inflamed gastrointestinal mucosa were used because they were known to be positive for iNOS. The IS RT-PCR technique localized iNOS mRNA to the same sites as immunoreactive iNOS in human gastritis associated with Helicobacter pylori infection, Crohn's disease, and experimental inflammatory bowel disease induced by the hapten TNBS, in rat colon and in guinea pig ileum. The detection of mRNA had an excellent signal-to-noise ratio. Preservation of tissue morphology was poorer than that with immunohistochemistry due the cycles of heating required in the PCR process. This method could be very useful in detecting iNOS gene expression in situations of excessive nonspecific staining with immunohistochemistry or of failure of antibodies to react because of species differences. This technique is also readily applicable to detect RNA and DNA markers of other disease processes.