Development of Real-Time PCR Assays for Detecting Matrix Metalloproteinases-2 & 9 Over-expression in Canine Mammary Tumours

Matrix Metalloproteinases (MMP) 2 & 9 are overexpressed in wide variety of cancers and play important roles in tumour cell proliferation, invasion and metastasis. Tissue and serum levels of MMP2 and MMP9 correlate with disease prognosis. Real-time PCR is emerging as an alternative or supplementary technique to immunohistochemistry (IHC) for sensitive detection of tumour markers in tissues. Therefore, in this study real-time PCR assays were standardized for detecting over-expression of MMP2 and MMP9 in canine mammary tumour (CMT). Primers designed for real-time PCR analysis of MMP2 & MMP9 were specific as indicated by amplification plots and meltcurve analysis of amplicons. These amplicons showed sharply defined melting curves with single peaks at expected melting points. The over-expression of MMP2 and MMP9 was examined in 12 CMT tissues by real-time PCR analysis. Over-expression of MMP2 & MMP9 was observed in 58.3(7/12) and 41.6 % (5/12) cases of CMT respectively. Expression levels of MMP2 were 2.23±0.10 to 29.80±3.5 fold higher and for MMP2, 1.5±0.56 to 22.67±2.35 fold higher in cases of CMT than normal mammary gland biopsy. Approximately 33.3 % (4/12) of CMT tissues showed co-expression of both MMP2 and MMP9. Further studies are required to correlate MMP2 and MMP9 expression levels with CMT prognosis.