PMID- 20947696
OWN - NLM
STAT- MEDLINE
DCOM- 20101221
LR  - 20190722
IS  - 1530-8561 (Electronic)
IS  - 0009-9147 (Linking)
VI  - 56
IP  - 12
DP  - 2010 Dec
TI  - 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.
PG  - 1845-53
LID - 10.1373/clinchem.2010.151233 [doi]
AB  - 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-mum 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.
FAU - Hennig, Guido
AU  - Hennig G
AD  - Siemens Healthcare Diagnostics Products, Molecular Research Germany, Cologne, 
      Germany.
FAU - Gehrmann, Mathias
AU  - Gehrmann M
FAU - Stropp, Udo
AU  - Stropp U
FAU - Brauch, Hiltrud
AU  - Brauch H
FAU - Fritz, Peter
AU  - Fritz P
FAU - Eichelbaum, Michel
AU  - Eichelbaum M
FAU - Schwab, Matthias
AU  - Schwab M
FAU - Schroth, Werner
AU  - Schroth W
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20101014
PL  - England
TA  - Clin Chem
JT  - Clinical chemistry
JID - 9421549
RN  - 0 (Biomarkers, Tumor)
RN  - 0 (Fixatives)
RN  - 0 (RNA, Messenger)
RN  - 1HG84L3525 (Formaldehyde)
RN  - 63231-63-0 (RNA)
RN  - 9007-49-2 (DNA)
SB  - IM
MH  - Automation, Laboratory
MH  - Biomarkers, Tumor/analysis
MH  - Breast Neoplasms/diagnosis/genetics/pathology
MH  - Cell Line, Tumor
MH  - DNA/*isolation & purification
MH  - Female
MH  - Fixatives
MH  - Formaldehyde
MH  - Gene Expression Profiling
MH  - Genotype
MH  - Humans
MH  - Paraffin Embedding
MH  - Polymerase Chain Reaction
MH  - *Polymorphism, Single Nucleotide
MH  - RNA/*isolation & purification
MH  - RNA, Messenger/*biosynthesis
MH  - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
MH  - Tissue Fixation
EDAT- 2010/10/16 06:00
MHDA- 2010/12/22 06:00
CRDT- 2010/10/16 06:00
PHST- 2010/10/16 06:00 [entrez]
PHST- 2010/10/16 06:00 [pubmed]
PHST- 2010/12/22 06:00 [medline]
AID - clinchem.2010.151233 [pii]
AID - 10.1373/clinchem.2010.151233 [doi]
PST - ppublish
SO  - Clin Chem. 2010 Dec;56(12):1845-53. doi: 10.1373/clinchem.2010.151233. Epub 2010 
      Oct 14.