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

摘要

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.