Preparation of formalin-fixed paraffin-embedded tissue cores for both RNA and DNA extraction

Palak G. Patel, Shamini Selvarajah, Suzanne Boursalie, Nathan E. How, Joshua Ejdelman, Karl Philippe Guerard, John M. Bartlett, Jacques Lapointe, Paul C. Park, John B A Okello, David M. Berman

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Article numbere54299
JournalJournal of Visualized Experiments
Volume2016
Issue number114
DOIs
StatePublished - Aug 21 2016
Externally publishedYes

Fingerprint

RNA
Formaldehyde
Paraffin
Paraffins
DNA
Tissue
Nucleic acids
Nucleic Acids
Digestion
Endopeptidase K
Organ Specificity
RNA-Directed DNA Polymerase
Methylation
DNA Methylation
Microarrays
Gene expression
Real-Time Polymerase Chain Reaction
Microscopes
Gene Expression
Polymerase Chain Reaction

Keywords

  • Archival
  • Basic protocol
  • Cancer
  • Cores
  • Deparaffinization
  • Extraction
  • FFPE
  • Issue 114
  • Nucleic Acids (DNA and RNA)
  • Pathology
  • Prostate
  • Tissue

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)

Cite this

Patel, P. G., Selvarajah, S., Boursalie, S., How, N. E., Ejdelman, J., Guerard, K. P., ... Berman, D. M. (2016). Preparation of formalin-fixed paraffin-embedded tissue cores for both RNA and DNA extraction. Journal of Visualized Experiments, 2016(114), [e54299]. https://doi.org/10.3791/54299

Preparation of formalin-fixed paraffin-embedded tissue cores for both RNA and DNA extraction. / Patel, Palak G.; Selvarajah, Shamini; Boursalie, Suzanne; How, Nathan E.; Ejdelman, Joshua; Guerard, Karl Philippe; Bartlett, John M.; Lapointe, Jacques; Park, Paul C.; Okello, John B A; Berman, David M.

In: Journal of Visualized Experiments, Vol. 2016, No. 114, e54299, 21.08.2016.

Research output: Contribution to journalArticle

Patel, PG, Selvarajah, S, Boursalie, S, How, NE, Ejdelman, J, Guerard, KP, Bartlett, JM, Lapointe, J, Park, PC, Okello, JBA & Berman, DM 2016, 'Preparation of formalin-fixed paraffin-embedded tissue cores for both RNA and DNA extraction', Journal of Visualized Experiments, vol. 2016, no. 114, e54299. https://doi.org/10.3791/54299
Patel PG, Selvarajah S, Boursalie S, How NE, Ejdelman J, Guerard KP et al. Preparation of formalin-fixed paraffin-embedded tissue cores for both RNA and DNA extraction. Journal of Visualized Experiments. 2016 Aug 21;2016(114). e54299. https://doi.org/10.3791/54299
Patel, Palak G. ; Selvarajah, Shamini ; Boursalie, Suzanne ; How, Nathan E. ; Ejdelman, Joshua ; Guerard, Karl Philippe ; Bartlett, John M. ; Lapointe, Jacques ; Park, Paul C. ; Okello, John B A ; Berman, David M. / Preparation of formalin-fixed paraffin-embedded tissue cores for both RNA and DNA extraction. In: Journal of Visualized Experiments. 2016 ; Vol. 2016, No. 114.
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