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Real-time PCR-based assay to quantify the relative amount of human and mouse tissue present in tumor xenografts.

BMC biotechnology (2011-12-20)
Sergio Y Alcoser, David J Kimmel, Suzanne D Borgel, John P Carter, Kelly M Dougherty, Melinda G Hollingshead
ABSTRACT

Xenograft samples used to test anti-cancer drug efficacies and toxicities in vivo contain an unknown mix of mouse and human cells. Evaluation of drug activity can be confounded by samples containing large amounts of contaminating mouse tissue. We have developed a real-time quantitative polymerase chain reaction (qPCR) assay using TaqMan technology to quantify the amount of mouse tissue that is incorporated into human xenograft samples. The forward and reverse primers bind to the same DNA sequence in the human and the mouse genome. Using a set of specially designed fluorescent probes provides species specificity. The linearity and sensitivity of the assay is evaluated using serial dilutions of single species and heterogeneous DNA mixtures. We examined many xenograft samples at various in vivo passages, finding a wide variety of human:mouse DNA ratios. This variation may be influenced by tumor type, number of serial passages in vivo, and even which part of the tumor was collected and used in the assay. This novel assay provides an accurate quantitative assessment of human and mouse content in xenograft tumors. This assay can be performed on aberrantly behaving human xenografts, samples used in bioinformatics studies, and periodically for tumor tissue frequently grown by serial passage in vivo.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Extract-N-Amp Tissue PCR Kit, sufficient for 1000 extractions, sufficient for 1000 amplifications

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