Technical Note: Comprehensive report on FFPE extraction methods

Key Points

  1. Shorter crosslink reversal incubation at higher temperature can replace longer incubation at lower temperautre with the FormaPure kit
  2. Higher-temperature crosslink reversal improves amplifiable DNA yield with the FormPaure kit
  3. Increased truXTRAC crosslink reversal time improves yield of amplifiable RNA and unique RNA molecules sequenced in FusionPlex Solid Tumor Assay
  4. Increased crosslink reversal time improves amplifiable RNA and DNA yield from tissue sections using Covaris truXTRAC
  5. Mineral oil functions as well as QIAGEN Deparaffinization Solution for deparaffinization with the QIAGEN AllPrep kit
  6. Increased crosslink reversal time improves amplifiable RNA and DNA yield with the Promega ReliaPrep extraction kit
  7. RNA and DNA yields using the Pinpoint kit are improved with increased crosslink reversal temperature
  8. Increasing crosslink reversal time substantially improves performance of the RecoverAll kit
  9. TRIzol can be used to isolate RNA from FFPE samples, but the recovery is reduced compared to the standard FormaPure extraction protocol
  10. Commercially available FFPE extraction kits result in variable library complexity

Abstract

Mutation detection based on DNA or RNA sequencing is becoming increasingly important in both human disease research and clinical settings. Typically, tissue biopsies are taken and stored as formalin-fixed, paraffin-embedded (FFPE) blocks, so that tissue architecture is maintained in a low-cost and low-tech manner that can be stored at room temperature for prolonged periods. However, nucleic acid extraction from FFPE samples produces poorer-quality material than extractions from freshly frozen material. Additionally, this material is often limited, and so gains in extraction efficiency as it relates to NGS library construction are highly valuable.

Therefore, we set out to evaluate ways in which we could improve the performance of six popular commercially available FFPE extraction kits as it relates to Anchored Multiplex PCR (AMP™) chemistry performance. Here we describe the protocol modifications we tested, identify the ones we found to be beneficial, and compare the performance of these kits to each other in the context of an Archer™ FusionPlex™ Assay.

Six different FFPE sample extraction kits and methods were optimized and their performance compared. The kits used were:

  • Agencourt Formapure Kit
  • QIAGEN® AllPrep® DNA/RNA FFPE Kit
  • Covaris truXTRAC FFPE RNA Kit
  • Ambion® RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE
  • Zymo Research Pinpoint™ Slide RNA Isolation System II
  • Promega ReliaPrep™ FFPE Total RNA Miniprep System
  • Ambion® TRIzol® Reagent

Key points from the FFPE extraction methods technical note

Shorter crosslink reversal incubation at higher temperature can replace longer incubation at lower temperature with the FormaPure kit

Shorter crosslink reversal incubation at higher temperature can replace longer incubation at lower temperature with the FormaPure kit

Higher temperature crosslink reversal improves amplifiable DNA yield with the FormaPure kit

Higher temperature crosslink reversal improves amplifiable DNA yield with the FormaPure kit

Increased truXTRAC crosslink reversal time improves yield of amplifiable RNA and unique RNA molecules sequenced in FusionPlex Solid Tumor Assay

Increased truXTRAC crosslink reversal time improves yield of amplifiable RNA and unique RNA molecules sequenced in FusionPlex Solid Tumor Assay

Increased crosslink reversal time improves amplifiable RNA and DNA yield from tissue sections using Covaris truXTRAC

Increased crosslink reversal time improves amplifiable RNA and DNA yield from tissue sections using Covaris truXTRAC

Mineral oil functions as well as QIAGEN Deparaffinization Solution for deparaffinization with the QIAGEN AllPrep kit

Mineral oil functions as well as QIAGEN Deparaffinization Solution for deparaffinization with the QIAGEN AllPrep kit

Increased crosslink reversal time improves amplifiable RNA and DNA yield with the Promega ReliaPrep extraction kit

Increased crosslink reversal time improves amplifiable RNA and DNA yield with the Promega ReliaPrep extraction kit

RNA and DNA yields using the Pinpoint kit are improved with increased crosslink reversal temperature

RNA and DNA yields using the Pinpoint kit are improved with increased crosslink reversal temperature

Increasing crosslink reversal time substantially improves performance of the RecoverAll kit

Increasing crosslink reversal time substantially improves performance of the RecoverAll kit

TRIzol can be used to isolate RNA from FFPE samples, but the recovery is reduced compared to the standard FormaPure extraction protocol

TRIzol can be used to isolate RNA from FFPE samples, but the recovery is reduced compared to the standard FormaPure extraction protocol

Commercially available FFPE extraction kits result in variable library complexity

Commercially available FFPE extraction kits result in variable library complexity

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For Research Use Only. Not for use in diagnostic procedures. For Research Use Only. Not for use in diagnostic procedures.