Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

[1]  E. Petricoin,et al.  Laser Capture Microdissection , 1996, Science.

[2]  N. S. Kumar,et al.  Coextraction and PCR Based Analysis of Nucleic Acids From Formalin‐Fixed Paraffin‐Embedded Specimens , 2015, Journal of clinical laboratory analysis.

[3]  M. Torrente,et al.  DNA extraction from formalin-fixed laryngeal biopsies: Comparison of techniques , 2011, Acta oto-laryngologica.

[4]  Jack A. Taylor,et al.  Potential for selection bias with tumor tissue retrieval in molecular epidemiology studies. , 2002, Annals of epidemiology.

[5]  G M Megson,et al.  Comparison of Techniques , 1999 .

[6]  D. Zack,et al.  Use of laser capture microdissection for analysis of retinal mRNA/miRNA expression and DNA methylation. , 2012, Methods in molecular biology.

[7]  M. Rubin,et al.  Targeted next-generation sequencing of advanced prostate cancer identifies potential therapeutic targets and disease heterogeneity. , 2013, European urology.

[8]  M. Monden,et al.  Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples. , 1999, Nucleic acids research.

[9]  M. Ferrer,et al.  Meeting report: discussions and preliminary findings on extracellular RNA measurement methods from laboratories in the NIH Extracellular RNA Communication Consortium , 2015, Journal of extracellular vesicles.

[10]  R. Klopfleisch,et al.  Excavation of a buried treasure--DNA, mRNA, miRNA and protein analysis in formalin fixed, paraffin embedded tissues. , 2011, Histology and histopathology.

[11]  Jeanne Kowalski,et al.  Hypermethylation of CpG Islands in Primary and Metastatic Human Prostate Cancer , 2004, Cancer Research.

[12]  H. Morreau,et al.  Assessment of a fully automated high-throughput DNA extraction method from formalin-fixed, paraffin-embedded tissue for KRAS, and BRAF somatic mutation analysis. , 2013, Experimental and molecular pathology.

[13]  Heike I. Grabsch,et al.  How to make tissue microarrays , 2009 .

[14]  J. Herman,et al.  Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Christian B. Woods,et al.  Analysis of repetitive element DNA methylation by MethyLight , 2005, Nucleic acids research.

[16]  W. Lam,et al.  DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses , 2011, Journal of visualized experiments : JoVE.

[17]  Brian Leyland-Jones,et al.  Optimization of RNA extraction from FFPE tissues for expression profiling in the DASL assay. , 2008, BioTechniques.

[18]  Serena Bonin,et al.  Nucleic acid extraction methods from fixed and paraffin-embedded tissues in cancer diagnostics , 2013, Expert review of molecular diagnostics.

[19]  G. Turashvili,et al.  Nucleic acid quantity and quality from paraffin blocks: defining optimal fixation, processing and DNA/RNA extraction techniques. , 2012, Experimental and molecular pathology.

[20]  Andrew H. Beck,et al.  Image-guided Coring for Large-scale Studies in Molecular Pathology , 2016, Applied immunohistochemistry & molecular morphology : AIMM.

[21]  A. Bossler,et al.  A simple and cost-effective method of DNA extraction from small formalin-fixed paraffin-embedded tissue for molecular oncologic testing , 2014, BMC Clinical Pathology.

[22]  S. Bonin,et al.  Multicentre validation study of nucleic acids extraction from FFPE tissues , 2010, Virchows Archiv.

[23]  Silke von Ahlfen,et al.  Determinants of RNA Quality from FFPE Samples , 2007, PloS one.

[24]  Hiltrud Brauch,et al.  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. , 2010, Clinical chemistry.

[25]  Scott E Kern,et al.  Why your new cancer biomarker may never work: recurrent patterns and remarkable diversity in biomarker failures. , 2012, Cancer research.

[26]  T. Schagat,et al.  Methods of RNA Quality Assessment , 2012 .

[27]  N. Sewankambo,et al.  Comparison of methods in the recovery of nucleic acids from archival formalin-fixed paraffin-embedded autopsy tissues. , 2010, Analytical biochemistry.