Automated High Throughput Nucleic Acid Purification From Formalin-Fixed Paraffin-Embedded Tissue Samples for Next Generation Sequence Analysis

PLoS One. 2017 Jun 1;12(6):e0178706. doi: 10.1371/journal.pone.0178706. eCollection 2017.

Abstract

Curation and storage of formalin-fixed, paraffin-embedded (FFPE) samples are standard procedures in hospital pathology laboratories around the world. Many thousands of such samples exist and could be used for next generation sequencing analysis. Retrospective analyses of such samples are important for identifying molecular correlates of carcinogenesis, treatment history and disease outcomes. Two major hurdles in using FFPE material for sequencing are the damaged nature of the nucleic acids and the labor-intensive nature of nucleic acid purification. These limitations and a number of other issues that span multiple steps from nucleic acid purification to library construction are addressed here. We optimized and automated a 96-well magnetic bead-based extraction protocol that can be scaled to large cohorts and is compatible with automation. Using sets of 32 and 91 individual FFPE samples respectively, we generated libraries from 100 ng of total RNA and DNA starting amounts with 95-100% success rate. The use of the resulting RNA in micro-RNA sequencing was also demonstrated. In addition to offering the potential of scalability and rapid throughput, the yield obtained with lower input requirements makes these methods applicable to clinical samples where tissue abundance is limiting.

MeSH terms

  • Automation*
  • DNA / genetics
  • DNA / isolation & purification*
  • Formaldehyde / chemistry*
  • High-Throughput Nucleotide Sequencing*
  • Paraffin Embedding*
  • RNA / genetics
  • RNA / isolation & purification*
  • Tissue Fixation / methods*

Substances

  • Formaldehyde
  • RNA
  • DNA

Grant support

This work was supported by the BC Cancer Foundation, and grants from Genome Canada/Genome British Columbia (Grant# 212SEQ/202SEQ), and the Canadian Institutes of Health Research (FDN-143288). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.