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https://hdl.handle.net/2440/63077
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Type: | Journal article |
Title: | Automated extraction of DNA and RNA from a single formalin-fixed paraffin-embedded tissue section for analysis of both single-nucleotide polymorphisms and mRNA expression |
Author: | Hennig, G. Gehrmann, M. Stropp, U. Brauch, H. Fritz, P. Eichelbaum, M. Schwab, M. Schroth, W. |
Citation: | Clinical Chemistry (Washington, DC): international journal of molecular diagnostics and laboratory medicine, 2010; 56(12):1845-1853 |
Publisher: | Amer Assoc Clinical Chemistry |
Issue Date: | 2010 |
ISSN: | 0009-9147 1530-8561 |
Statement of Responsibility: | Guido Hennig, Mathias Gehrmann, Udo Stropp, Hiltrud Brauch, Peter Fritz, Michel Eichelbaum, Matthias Schwab, and Werner Schroth |
Abstract: | BACKGROUND: There is an increasing need for the identification of both DNA and RNA biomarkers from pathodiagnostic formalin-fixed paraffin-embedded (FFPE) tissue samples for the exploration of individualized therapy strategies in cancer. We investigated a fully automated, xylene-free nucleic acid extraction method for the simultaneous analysis of RNA and DNA biomarkers related to breast cancer. METHODS: We copurified both RNA and DNA from a single 10-µm section of 210 paired samples of FFPE tumor and adjacent normal tissues (1–25 years of archival time) using a fully automated extraction method. Half of the eluate was DNase I digested for mRNA expression analysis performed by using reverse-transcription quantitative PCR for the genes estrogen receptor 1 (ESR1), progesterone receptor (PGR), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) (ERBB2), epoxide hydrolase 1 (EPHX1), baculoviral IAP repeat-containing 5 (BIRC5), matrix metallopeptidase 7 (MMP7), vascular endothelial growth factor A (VEGFA), and topoisomerase (DNA) II alpha 170kDa (TOP2A). The remaining undigested aliquot was used for the analysis of 7 single-nucleotide polymorphisms (SNPs) by MALDI-TOF mass spectrometry. RESULTS: In 208 of 210 samples (99.0%) the protocol yielded robust quantification-cycle values for both RNA and DNA normalization. Expression of the 8 breast cancer genes was detected in 81%–100% of tumor tissues and 21%–100% of normal tissues. The 7 SNPs were successfully genotyped in 91%–97% of tumor and 94%–97% of normal tissues. Allele concordance between tumor and normal tissue was 98.9%–99.5%. CONCLUSIONS: This fully automated process allowed an efficient simultaneous extraction of both RNA and DNA from a single FFPE section and subsequent dual analysis of selected genes. High gene expression and genotyping detection rates demonstrate the feasibility of molecular profiling from limited archival patient samples. |
Keywords: | Cell Line, Tumor Humans Breast Neoplasms Formaldehyde DNA RNA RNA, Messenger Fixatives Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Paraffin Embedding Tissue Fixation Gene Expression Profiling Polymerase Chain Reaction Genotype Polymorphism, Single Nucleotide Female Automation, Laboratory Biomarkers, Tumor |
Rights: | Copyright © 2010 by the American Association for Clinical Chemistry. |
DOI: | 10.1373/clinchem.2010.151233 |
Published version: | http://dx.doi.org/10.1373/clinchem.2010.151233 |
Appears in Collections: | Aurora harvest Pharmacology publications |
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