Optimization of methods to assess mitochondrial DNA in archival paraffin-embedded tissues from mammary canine tumors

Abstract

In this study we describe the alterations used to extract and amplify mitochondrial desoxyribonucleic acid (DNA) from formalin-fixed paraffin-embedded samples of canine mammary tumors. The epithelial and mesenchymal components (chondromyxoid and chondroid) of each tumor, as well as the normal mammary gland tissues, were manually microdissected from 19 mixed canine mammary tumors (10 benign mixed tumors and nine carcinomas arising in mixed tumors). DNA was extracted by Invisorb® Spin Tissue Mini Kit, with protocol changes proposed by the manufacturer. A 273-bp fragment was amplified by polymerase chain reaction (PCR) and submitted to automatic sequence analysis. The fragment was successfully analyzed in 100% of the samples. However, an additional lysis step, the reduction of volume in buffer solutions and PCR, a higher annealing temperature and an increase in the number of PCR cycles were required. The initial PCR products were diluted and re-amplified in six samples so that they could be successfully analyzed.