Quantitation of bovine cytokine mRNA in milk cells of healthy cattle by real-time TaqMan ® polymerase chain reaction

Abstract

Here we present a novel methodology to quantitate bovine cytokines and growth factors contributing to immunity against bacterial infections of the mammary gland in cattle. Real-time TaqMan ® PCR systems were developed to overcome limitations of conventional quantitative PCR methods. The TaqMan ® method is based on the cleavage of fluorescent dye-labeled probes by the 5′–3′ exonuclease activity of the Taq DNA polymerase during PCR and measurement of fluorescence intensity by an automated spectrophotometer integrated in a sequence detection system (Applied Biosystems, Foster City, CA). The bovine-specific TaqMan ® probes were designed to encompass an intron, thus allowing differentiation between complementary DNA (cDNA) and genomic DNA (gDNA) amplification products. Quantitative analysis of cytokine cDNA was performed in comparison to bovine glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Messenger RNA (mRNA) from the universally expressed housekeeping gene GAPDH proved to be useful as an amplification control and allowed for correction of variations in different numbers of cells in the starting material, in the efficiencies of RNA extraction and reverse transcription. With this method, high-throughput analysis of large numbers of samples was possible within a short time. In addition, decreasing the numbers of working steps shortened the time for analysis and increased accuracy. Profiles of cytokines (interleukin (IL)-2, IL-6, IL-8, IL-12 p40, TNF-α, IFN-γ) and granulocyte–macrophage colony stimulating factor (GM-CSF) were established in normal lactating cattle. Differences of cytokine profiles obtained with the real-time TaqMan ® PCR system and conventional methods are discussed.