Expression profile of Toll-like receptor 2 gene in dairy buffaloes with mastitis

Toll-like receptors (TLRs) are a group of protein essential for the detection of pathogens and the initiation of immune response. Thus, the aim of this study was to evaluate the relative expression of Toll-like receptor 2 (TLR-2) gene in milk cells of dairy buffaloes with mastitis. Milk samples of 10 animals with mastitis and 10 animals without mastitis was used for RNA extraction by Trizol[R] reagent (Invitrogen, USA). The verification of mastitis was performed by California Mastitis Test and somatic cell count. The RNA quality and concentration were measured by spectrophotometer and the absence of contamination by genomic DNA was confirmed in a Qubit[R] 2.0 fluorometer (Invitrogen, USA). The cDNA was amplified by quantitative real-time PCR using specific primers. RPLP0 primer were obtained in the literature, while TLR-2 and EEF1A1 primers were described on the literature to bovine studies and redesigned from the homologous buffalo mRNA. The EEF1A1 and RPLP0 genes were tested by the geNorm program and used as endogenous controls for normalization of TLR-2 gene expression. Data were analyzed using the PROC MIXED of Statistical Analysis System (SAS Institute Inc., Cary, NC). The [DELTA][DELTA]Ct was estimated using orthogonal contrasts between treatments and endogenous controls and target genes. Values were considered significant when the approximation of [DELTA][DELTA]Ct showed p