Quantitation of equine cytokine mRNA expression by reverse transcription-competitive polymerase chain reaction.

The research article presents the development of a method called reverse transcription-competitive polymerase chain reaction (RT-cPCR) for measuring specific immune response proteins (cytokine mRNA expression) in horses.

Method Development

• The technique was designed to measure mRNA expression of several different types of cytokines – biochemical signaling molecules that mediate and regulate the immune system and inflammation responses, namely interleukin (IL)-1alpha, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 p35, IL-12 p40, interferon-gamma (INF-gamma), and tumor necrosis factor-alpha (TNF-alpha).
• To detect these cytokines, the team used unique sequencing primers to stimulate peripheral blood mononuclear cells with a substance called concanavalin A. The amplified products’ specificity was confirmed by performing sequencing.

Quantitative Procedure

• For quantitative assay, the researchers created competitor DNA fragments (also known as a functionally similar mimic) which had the same primer template as a horse cytokine but were of different sizes to distinguish them on a gel.
• These dilutions of mimic were introduced into PCR reactions that contained a consistent amount of horse cDNA. After gel electrophoresis and staining, a densitometric analysis was performed on the bands that corresponded to the target and the mimic, resulting in a standard curve from which the target cDNA amount was inferred.

Result Analysis and Implications

• The team carried out several tests for IL-6 gene expression on different days, yielding a coefficient of variation of 6.6%. The results were then normalized to beta-actin mRNA expression to reduce variation caused by differences in the efficiency of reverse transcription, or by discrepancies in the quantity or quality of the original RNA. Beta-actin mRNA expression was used as a standard, or ‘house-keeping gene’ as it is consistently expressed.
• The method allowed the detection of minor differences (2-fold) in cytokine mRNA expression, thus highlighting its sensitivity and reliability.
• The authors conclude that this method could be beneficial in tracking changes within horse cytokine gene expression, both in the lab (in vitro) and in living organisms (in vivo). They also suggest the potential broad applicability of the RT-cPCR method in quantifying cytokine gene expression across different animal species, hence, holding veterinary significance.