Development of a competitive reverse transcription - polymerase chain reaction assay for quantitation of equine interferon gamma, interleukin-2, interleukin-4 and glyceraldehyde-3-phosphate dehydrogenase

Interferon gamma (IFN-gamma), interleukin-2 (IL-2) and interleukin-4 (IL-4) are important as immunological mediators which are characteristic of T-helper 1 (Th1) or T-helper 2 (Th2) responses. An assay was developed to quantify equine cytokines IFN-gamma, IL-2, and IL-4 and the metabolic protein glyceraldehyde-3-phosphate dehydrogenase (G3PDH) by reverse transcription - polymerase chain reaction (RT-PCR). Competitive RNA (cRNA) fragments for employment in the RT-PCR assay were generated by recombinant PCR as deletion mutants of target sequences. Optimal methodology was determined for the following: production of cRNA pieces; reverse transcription with particular emphasis on RT primers; PCR amplification, including optimization of buffer composition, primers, number of amplification cycles and hot start procedures; electrophoresis, including preparation of low melting temperature agarose gels and densitometric scanning of bands; and analysis of data, including a linear regression of plotted fluorescence measurements and a method of iteration for quantification of target sequences.; A computer program was developed to import data files generated from densitometric analysis of samples after electrophoresis. The computer program was designed to detect and identify bands and store data in a database linking all information relating to each sample for subsequent retrieval and analysis of data.; The competitive RT-PCR assay and computer program were used in in-vitro and in-vivo stimulation studies of IFN-gamma, IL-2, IL-4 and GPM transcript production by equine peripheral blood mononuclear cells (PBMC's). IL-4 transcripts were found to be extremely rare and difficult to quantify; whereas, IFN-gamma and IL-2 could be quantified in PBMC's stimulated with Concanavalin-A. G3PDH was determined to be acceptable as a housekeeping gene for equine PBMC's as transcript levels were directly related to the number of PBMC's in a sample, or to the amount of total or messenger RNA in a sample. Stimulation of PBMC's did not compromise the usefulness of G3PDH as a housekeeping gene. There are indications from the results that the capacity of PBMC's to produce IFN-gamma and IL-2 transcripts increases following exposure of a horse to a modified live vaccine against Equine Viral Arteritis (EVA), but decreases when a horse is exposed to a virulent strain of Equine Arteritis Virus (EAV).