Formalin fixed paraffin embedded RNA extraction, a double-edged sword?

There’s no doubt that formalin fixation is a great way to preserve and archive samples in a cost-effective and compact manner, especially compared to methods like bulky and expensive freezers. However, a major disadvantage of formalin fixation is the large amount of nucleic acid degradation in samples so treated, especially RNA. That being said, gene expression analysis is still possible with careful preparation of these types of samples and ARQ Genetics has many years of experience in handling them and getting reproducible results via qPCR. The following is a brief overview of various methods for preparing RNA from formalin fixed samples.

RNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues presents unique challenges but can be achieved effectively using specialized protocols. Here’s a comprehensive overview of methods and considerations:

Key Extraction Methods

Spin Column-Based Kits 

  • Uses silica membrane technology with optimized lysis buffers to reverse formaldehyde-induced crosslinks[1]
  • Features:
    • 15-minute proteinase K digestion followed by 80°C incubation to reverse modifications[1]
    • Integrated DNase treatment to remove genomic DNA[1]
    • Compatible with automation
    • Processes 1-4 tissue sections (5-10 µm thick) in 70 minutes[1]

Paramagnetic Bead Systems 

  • Employs SPRI bead chemistry for challenging FFPE samples[2]
  • Advantages:
    • Handles 10+ year-old samples[2]
    • Scalable for high-throughput workflows[2]
    • Maintains RNA integrity better than traditional methods[2]

Optimized Protocol Considerations

  • Enzymatic proteolysis followed by glass-fiber purification helps recover fragmented RNA[3]
  • Heating steps (80-100°C) help reverse formaldehyde adducts[3][4]
  • Specialized lysis buffers prevent further RNA degradation during processing[1]

Major Challenges

  • RNA Fragmentation: Average fragment size typically <200 nucleotides due to fixation[4][5]
  • Chemical Modifications:
    • Methylol adducts and crosslinks impair enzymatic reactions[3][1]
    • Damage increases with sample age[3]
  • Yield Variability: Affected by:
    • Fixation duration (24-72 hours optimal)[6][7]
    • Section thickness (10-20 µm recommended)[7]
    • Tissue cellularity[7]

Best Practices

  1. Quality Control:
    • Use DV200 metric (percentage of RNA >200 nucleotides) for integrity assessment[5]
    • Prioritize samples with >50% RNA fragments >200 nt for sequencing[5]
  2. Downstream Optimization:
    • Design small amplicons (<100 bp) for PCR/qPCR[4][1]
    • Use random hexamers instead of oligo-dT for cDNA synthesis[1]
    • Consider RNA enrichment kits for NGS applications[5]
  3. Troubleshooting:
    • For low yields: Increase section thickness or quantity[7]
    • For DNA contamination: Use booster buffers with DNase[1]
    • For failed extractions: Use FFPE as backup when fresh-frozen samples are unavailable[6]

Leading commercial kits demonstrate comparable performance for qPCR applications when using samples fixed <10 years, with success rates >90% for gene expression profiling when following optimized protocols[6][1][5]. Recent advancements in probe-based enrichment technologies now enable reliable analysis of even highly degraded RNA[5].

 

  1. https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/rna-purification/total-rna/rneasy-ffpe-kit
  2. https://www.beckman.com/reagents/genomic/rna-isolation/from-ffpe-tissue
  3. https://www.thermofisher.com/us/en/home/references/ambion-tech-support/rna-isolation/general-articles/extraction-of-nucleic-acids-from-ffpe-samples.html
  4. https://assets.thermofisher.com/TFS-Assets/LSG/brochures/cms_053192.pdf
  5. https://www.illumina.com/content/dam/illumina-marketing/documents/products/technotes/evaluating-rna-quality-from-ffpe-samples-technical-note-470-2014-001.pdf
  6. https://pmc.ncbi.nlm.nih.gov/articles/PMC8143498/
  7. https://nanostring.com/wp-content/uploads/MAN-10050-05-Preparing-RNA-from-FFPE-Samples.pdf