Maximize success with FFPE and other challenging cancer sample types

Fine-tuning FFPE sample preparation

Fixation in formalin-based solutions and embedding in paraffin wax to make FFPE tissue blocks is the gold standard for tissue preservation. FFPE tissue sections, therefore, represent a treasure trove of cancer biomarker information. However, FFPE tissues are notoriously tricky to work with, creating a need for specialized tools and technologies for the molecular analysis of FFPE samples. To accurately attribute the changes caused by cancer on a molecular level depends on the ability to compare normal tissue with tissue representing different stages of disease. This requires retrieving nucleic acids of the highest integrity.

What are the key challenges you’re confronted with?

• Cross-linking: Formalin fixation causes a chemical reaction between formaldehyde and nucleic acids, resulting in crosslinking with proteins and other biomolecules and impacting downstream applications.
• Cytosine deamination: This occurs randomly during formalin fixation and, with aging, turns cytosine into a Uracil. This has implications in downstream DNA sequencing, manifesting as a C>T|G>A transition.
• Nucleic acid fragmentation: Attributed to the fixation process, aging, high temperatures during the embedding process, oxidation and staining.
• Low yields: Crosslinking and chemical modifications such as oxidation and the thickness of the FFPE sections can contribute to this.
• Poor quality: Crosslinking and chemical modifications all impact the integrity of nucleic acids.

Tips & tricks for successful nucleic acid isolation from FFPE samples

DNA and RNA isolation from FFPE samples can require hours of optimization. Because FFPE samples are often limited and therefore, precious, you often don’t have enough sample for multiple re-runs and trial-and-error attempts at nucleic acid isolation.

Sometimes, with FFPE samples, you simply don’t get a second chance.

We can help you get it right from the start. Access our technical guidelines for all the do’s & don’ts and best practices when it comes to FFPE sample prep.

Norbert Hochstein: Methylation analysis by Pyrosequencing – impact of amplicon length and DNA input quantity

Norbert gives an overview of a Pyrosequencing workflow for methylation analysis followed by key findings of his Pyrosequencing study using FFPE samples and the impacts of amplicon length and DNA input quantity on results.

Jonathan Shaffer: Whole transcriptome NGS of FFPE samples

Jon introduces a new technology to efficiently remove rRNA from FFPE samples during whole transcriptome RNA-seq that dramatically increases the reads mapped to genes and detected genes, leading to robust differential expression analysis.

Jonathan Shaffer: Multimodal NGS analysis from FFPE samples

Jon talks about a new workflow – multimodal NGS – for simultaneous profiling of both DNA variants and RNA fusions. He will discuss, with examples, how this solution allows consolidated DNA and RNA targeted library preparation from FFPE samples.

Leif Schauser: NGS data analysis using QIAGEN CLC Genomics Workbench

Leif introduces the QIAGEN CLC Genomics Workbench for NGS data analysis and the key features that are relevant to FFPE. In a live demo, he will guide you through the key steps of DNA-seq and RNA-seq data analysis workflows.