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 Guido Hennig, 1,2 Mathias Gehrmann, 1 Udo Stropp, 1 Hiltrud Brauch, 3 Peter Fritz, 3 Michel Eichelbaum, 3 Matthias Schwab, 3,4 and Werner Schroth 3* BACKGROUND: There is an increasing need for the iden- tification of both DNA and RNA biomarkers from pathodiagnostic formalin-fixed paraffin-embedded (FFPE) tissue samples for the exploration of individu- alized 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-m section of 210 paired samples of FFPE tumor and adjacent normal tissues (1–25 years of ar- chival 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 onco- gene 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 topoisom- erase (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 tu- mor 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 concor- dance 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. © 2010 American Association for Clinical Chemistry The identification and validation of molecular markers in formalin-fixed paraffin-embedded (FFPE) 5 tissue has been a field of intensive research in recent years because expression profiling, geno- typing, and mutation analysis have been increasingly shown to aid in cancer diagnosis and to guide cancer treatment. As yet, however, only a limited number of marker panels or individual markers developed from histopathological tissue specimens have been intro- duced into routine clinical practice [e.g., On- cotypeDX®: NCCN Clinical Practice Guidelines in Oncology (1 ) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) 6 mutation analysis for 1 Siemens Healthcare Diagnostics Products, Molecular Research Germany, Cologne, Ger- many; 2 Siemens Healthcare Diagnostics, Eschborn, Germany; 3 Dr. Margarete Fischer- Bosch-Institute of Clinical Pharmacology, Stuttgart, and University Tu ¨ bingen, Germany; 4 Department of Clinical Pharmacology, University Hospital Tu ¨ bingen, Germany. * Address correspondence to this author at: Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Auerbachstrasse 112, 70376 Stuttgart, Germany. Fax +49-711-859295; e-mail werner.schroth@ikp-stuttgart.de. Received May 28, 2010; accepted September 20, 2010. Previously published online at DOI: 10.1373/clinchem.2010.151233 5 Nonstandard abbreviations: FFPE, formalin-fixed, paraffin-embedded; SNP, single-nucleotide polymorphism; GOI, genes of interest; MS, mass spectrometry; HWE, Hardy-Weinberg equilibrium; RT-qPCR, reverse transcription– quantitative PCR; Cq, quantification cycle; MAF, minor allele frequency. 6 Human genes: KRAS, v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; ESR1, estrogen receptor 1; PGR, progesterone receptor; ERBB2, v-erb-b2 erythroblastic leuke- mia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian); EPHX1, epoxide hydrolase 1; BIRC5, baculoviral IAP repeat-containing 5; MMP7, matrix metallopeptidase 7; VEGFA, vascular endothelial growth factor A; TOP2A, topoisomer- ase (DNA) II alpha 170kDa; RPL37A, ribosomal protein L37a; PAEP, progestagen- associated endometrial protein; ESR2, estrogen receptor 2; NCOA3, nuclear receptor coactivator 3; PPARGC1B, peroxisome proliferator-activated receptor gamma, coactiva- tor 1 beta; RRAS2, related RAS viral (r-ras) oncogene homolog 2; CYP2D6, cytochrome P450, family 2, subfamily D, polypeptide 6; HOXB13, homeobox B13; IL17RB, interleukin 17 receptor B. Clinical Chemistry 56:12 1845–1853 (2010) Automation and Analytical Techniques 1845 Downloaded from https://academic.oup.com/clinchem/article/56/12/1845/5622202 by guest on 19 June 2022