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|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|
|Citation:||Clinical Chemistry, 2010; 56(12):1845-1853|
|Publisher:||Amer Assoc Clinical Chemistry|
|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.|
|Appears in Collections:||Pharmacology publications|
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