Quantitative measurement of equine cytokine mRNA expression by polymerase chain reaction using target-specific standard curves

Abstract Quantification of cytokine mRNA using reverse transcription coupled with the polymerase chain reaction (RT-PCR) has become a corner stone of the study of cytokine regulation. Quantitative competitive RT-PCR (QCRT-PCR) is commonly accepted as a reliable method for quantifying differences in mRNA levels but is both labor- and reagent-intensive. A noncompetitive polymerase chain reaction method that utilizes cytokine-specific, plasmid-derived, standard curves was developed for the quantification of equine cytokine mRNA. The assay can be performed on minute samples of cellular material, utilizes sequences identical to wild-type for the generation of standard curves, is technically facile, less reagent- and labor-intensive than competitive methods, easily accommodates high sample throughput without the use of radioactive labels, and generates replicate samples to allow statistical analysis of the data. We demonstrate the utility of the assay, which is easily adapted to any cloned mRNA sequence, using equine interleukin-10 (IL-10). Both IL-10 and β-actin cDNA were amplified in triplicate PCR reactions from oligo-dT primed RT reactions. Dilutions of plasmid DNA encoding the respective sequence, equine IL-10 or β-actin, were also amplified in triplicate reactions in the same run. β-actin cycling parameters were modified to maintain the amplification in its exponential phase by decreasing both cycle number and cDNA volume relative to the parameters used for cytokine amplification. Following amplification, aliquots of the PCR reactions were hybridized with sequence-specific tris (2,2′-bipyridine) ruthenium II chelate labeled oligonucleotide probes and quantified using the QPCR System 5000. Plasmid derived values were used to generate a standard curve for the interpolation of mRNA content in unknown cDNA samples. β-actin values were used to derive a factor for the relative normalization of differences among cDNA samples that are inherent in the RNA extraction and RT steps. This assay resolves at least 2-fold differences in message, is reproducible, and has a dynamic range on the order of 3 logs.