How to Apply NGS Technologies to FFPE Tissues?

Formalin-fixed and paraffin-embedded (FFPE) tissues represent a treasure trove of biological material, often derived from archival specimens with accompanying clinical data. However, the formalin fixation and paraffin embedding processes can lead to challenges in nucleic acid extraction and subsequent analysis. Leveraging next-generation sequencing (NGS) technologies present a breakthrough in overcoming these obstacles, enabling comprehensive genomic and transcriptomic profiling of FFPE samples with high accuracy and precision.

Procedures for Extracting Nucleic Acids from FFPE Tissues

The process of extracting nucleic acids from FFPE tissues is a crucial step in genomic and transcriptomic analysis. The formalin fixation and paraffin embedding of tissue samples can lead to cross-linking, fragmentation, and degradation of nucleic acids, presenting significant challenges for extraction. However, innovative procedures have been developed to overcome these challenges and extract high-quality DNA and RNA from FFPE tissues.

• Sample preparation. To obtain the tissue sections, locate and mark the regions of interest on the FFPE tissue block or slides. If using slides, deparaffinize the tissue sections by immersing them in xylene or a xylene substitute for a few minutes. Then, rehydrate the sections using a series of graded alcohols (e.g., 100%, 95%, 70% ethanol). Finally, briefly air-dry the slides or blocks to remove residual alcohol.
• Tissue digestion. The buffer composition may vary depending on the specific protocol or commercial kit being used. To prepare a digestion buffer with proteinase K, apply the buffer to the tissue sections or scrape the tissue from the slides into a sterile tube containing the digestion buffer. Incubate the samples at an appropriate temperature (e.g., 55-60°C) for several hours overnight to allow proteinase K to digest the tissue and release nucleic acids.
• De-crosslinking. After digestion, inactivate the proteinase K by heating the samples at a higher temperature (e.g., 90-95°C) for a short period (e.g., 10-15 minutes). This step helps to reverse the cross-linking caused by formalin fixation and promotes the release of nucleic acids.
• Nucleic acid purification. Transfer the lysate to a clean tube and proceed with nucleic acid purification. Commercial kits specifically designed for FFPE samples are available and provide detailed protocols for purification. These kits often use spin columns or magnetic bead-based systems to isolate and purify nucleic acids (DNA or RNA) from the lysate.

Decoding Genomes with NGS Technologies

NGS technologies have revolutionized genomic analysis, providing unparalleled insights into genetic variations, gene expression, and genomic architecture. However, due to the fragmented and relatively poor quality of nucleic acids extracted from FFPE samples, long-read sequencing platforms are generally incompatible with data obtained from FFPE tissue. Therefore, there is a need for more effective methods to extract genetic material from FFPE tissue and for downstream sequencing.

Significances of NGS Technologies for FFPE Tissues

• Preservation of archival samples. FFPE tissues are commonly used in clinical and research settings because they represent a vast collection of preserved samples over extended periods. NGS allows researchers to leverage these valuable archival samples and extract valuable genomic and transcriptomic information from them.
• Genome-wide analysis. NGS enables comprehensive, high-throughput analysis of the entire genome or specific regions of interest. It allows for the identification of genetic variants, including single nucleotide variations (SNVs), insertions/deletions (indels), copy number variations (CNVs), and structural rearrangements, providing insights into the genomic landscape of FFPE tissues.
• Mutation profiling and biomarker discovery. NGS enables the detection of somatic mutations and genomic alterations in FFPE tissues. By comparing tumor and normal tissue samples, researchers can identify driver mutations, oncogenic pathways, and potential therapeutic targets. NGS can also aid in the discovery of predictive biomarkers for treatment response or prognosis.

Creative Bioarray Relevant Recommendations

Creative Bioarray provides a range of normal and diseased tissue blocks. Our high-quality tissues are banked under strict collection protocols to ensure that both paraffin-embedded and frozen tissues are all well-characterized, clinically annotated tissues needed for critical experiments.

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