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Research Article

Quantitative and Sensitive Detection of Cancer Genome Amplifications from Formalin Fixed Paraffin Embedded Tumors with Droplet Digital PCR

Lincoln Nadauld1#, John F Regan2#, Laura Miotke1, Reet K Pai3, Teri A Longacre3, Shirley S Kwok3, Serge Saxonov2, James M Ford1 and Hanlee P Ji14*

1Division of Oncology, Department of Medicine, Stanford University School of Medicine, Stanford, CA,USA

2Bio-Rad, Inc., Pleasanton, CA, USA

3Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA

4Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA

#These authors have contributed equally to this work

*Corresponding Author:
Hanlee P Ji
Division of Oncology, Department of Medicine
Stanford University School of Medicine
CCSR 1115, 269 Campus Drive, Stanford
CA 94305-5151, USA
Tel: 650-721-1503
Fax: 650-725-1420
E-mail: [email protected]

Received Date: August 22, 2012; Accepted Date: September 25, 2012; Published Date: September 28, 2012

Citation: Nadauld L, Regan JF, Miotke L, Pai RK, Longacre TA, et al. (2012) Quantitative and Sensitive Detection of Cancer Genome Amplifications from Formalin Fixed Paraffin Embedded Tumors with Droplet Digital PCR. Transl Med 2:107. doi:10.4172/2161-1025.1000107

Copyright: © 2012 Nadauld L, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

For the analysis of cancer, there is great interest in rapid and accurate detection of cancer genome amplifications containing oncogenes that are potential therapeutic targets. The vast majority of cancer tissue samples are formalin fixed and paraffin embedded (FFPE) which enables histopathological examination and long term archiving. However, FFPE cancer genomic DNA is often degraded and generally a poor substrate for many molecular biology assays. To overcome the issues of poor DNA quality from FFPE samples and detect oncogenic copy number amplifications with high accuracy and sensitivity, we developed a novel approach. Our assay requires nanogram amounts of genomic DNA, thus facilitating study of small amounts of clinical samples. using droplet digital PCR (ddPCR), we can determine the relative copy number of specific genomic loci even in the presence of intermingled normal tissue. We used a control dilution series to determine the limits of detection for the ddPCR assay and report its improved sensitivity on minimal amounts of DNA compared to standard. Real-Time PCR. To develop this approach, we designed an assay for the fibroblast growth factor receptor 2 genes (FGFR2) that is amplified in gastric and breast cancers as well as others. We successfully utilized ddPCR to ascertain FGFR2 amplifications from FFPE-preserved gastrointestinal adenocarcinomas.

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