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.
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.
Siemens Healthcare Diagnostics Products, Molecular Research Germany, Cologne, Germany.
Guido
G
Hennig
Mathias
M
Gehrmann
Udo
U
Stropp
Hiltrud
H
Brauch
Peter
P
Fritz
Michel
M
Eichelbaum
Matthias
M
Schwab
Werner
W
Schroth
D057205:Automation, Laboratory; D014408:Biomarkers, Tumor / Q000032:analysis; D001943:Breast Neoplasms / Q000175:diagnosis / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D004247:DNA / Q000302:isolation & purification*; D005260:Female; D005404:Fixatives; D005557:Formaldehyde; D020869:Gene Expression Profiling; D005838:Genotype; D006801:Humans; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D012313:RNA / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000096:biosynthesis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D016707:Tissue Fixation
D014408:Biomarkers, Tumor; D005404:Fixatives; D012333:RNA, Messenger; D005557:Formaldehyde; D012313:RNA; D004247:DNA