Determination of internal control for gene expression studies in equine tissues and cell culture using quantitative RT-PCR.

Quantitative reverse transcription polymerase chain reaction (RT-PCR) has become a basic, reliable and sensitive modern technique, in both biological research and clinical diagnosis, for investigation of gene expression and validation of cDNA microarray analysis. Accurate mRNA quantification using quantitative RT-PCR commonly requires data normalization through stable housekeeping genes (HKGs). Selection of HKGs for data normalization is critical for accurate mRNA quantification. Our objective was to evaluate a set of candidate HKGs as internal controls for gene expression studies using quantitative RT-PCR in equine tissues and cell culture. One-step quantitative RT-PCR for 6 HKGs was performed using total RNA from equine tissue samples and cultured peripheral blood mononuclear cells (PBMCs). The stability of HKGs was mainly evaluated by analysis of variance, analyses of the standard deviation and coefficient of variation of Ct, and change of Ct of HKGs between control and treated samples. 18S rRNA consistently showed the smallest standard deviation and coefficient of variation, and the least change of Ct between control and treated samples, thus was identified as the most stable HKG for mRNA data normalization in quantitative RT-PCR for studying gene expression in equine tissues and cultured PBMCs.

Molecular cloning and characterization of equine Toll-like receptor 9.

Innate immunity relies on a series of germline-encoded pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), to detect conserved microbial components. TLR9 is typically expressed intracellularly in immune cells such as dendritic cells and recognizes unmethylated bacterial or viral cytosine-phosphate-guanine DNA (CpG-DNA). To investigate innate immune responses through TLR9 signaling pathway in horses, we cloned and characterized equine TLR9. Protein sequence analysis shows that equine TLR9 has a typically conserved cytosolic Toll/interleukin-1 receptor (TIR) domain, three leucine-rich repeat (LRR) motifs, with greater than 82% identity to human, monkey, bovine, canine, feline, porcine and ovine orthologs. Equine TLR9 mRNA expression was characterized for spleen, lymph node, and peripheral blood leukocyte samples. Flow cytometric analysis of equine TLR9 expression using a cross-reactive TLR9 mAb identified high constitutive expression of equine TLR9 in PMNs, CD4(+) and CD8(+) T-lymphocytes as well as other leukocytes; similar to human TLR9 expression. The conservation of equine TLR9 and high expression profile in leukocytes suggests that equine TLR9 is a frequent target for unmethylated CpG-DNA, an essential mechanism for the activation of innate immunity.