RNA Sequencing Solutions for FFPE Cancer Tissue Samples

RNA sequencing is essential for understanding cancer on a molecular level. But when it comes to FFPE (formalin-fixed paraffin-embedded) tissue samples, often the only ones left from older studies, the challenge becomes clear. These samples tend to have fragmented RNA, making sequencing much harder.

Thankfully, new advancements are making it possible to work with these degraded samples. Fresh techniques are allowing researchers to extract RNA from FFPE tissues with far more accuracy, opening doors to new cancer insights. For many, these FFPE samples are the only ones available, so improving how we analyze them is a game-changer.

In this blog, we’ll look at how recent innovations in RNA sequencing are helping overcome the challenges of working with FFPE cancer tissues.

What are FFPE Tissue Samples?

FFPE (formalin-fixed paraffin-embedded) tissue samples are biological specimens that have been preserved through a process of fixation with formalin and embedding in paraffin wax. This method stabilizes the tissue, preserving its cellular structure and molecular integrity for long-term storage.

These archived samples are often the only available resource for retrospective studies on cancer progression, genetic mutations, and treatment responses. In addition, FFPE blocks are routinely used for diagnostic purposes, such as immunohistochemistry and DNA sequencing. 

In cancer research, FFPE samples are crucial because they retain nucleic acids but with significant degradation and chemical modifications that impact sequencing quality, making them valuable for various molecular analyses, including RNA sequencing.

Innovations in RNA Sequencing for FFPE Cancer Tissues

Recent innovations in RNA sequencing are making it possible to extract meaningful data from FFPE cancer tissues, which were previously challenging due to RNA degradation and crosslinking. Here are some key advancements:

1. Improved RNA Extraction Techniques

Process: FFPE tissues are known for their chemical crosslinking and fragmentation, which complicate the extraction of high-quality RNA. Traditional RNA extraction methods often fail to yield sufficient, intact RNA from these samples.

Recent Advancements: New RNA extraction kits and protocols from Qiagen, Thermo Fisher, and others are indeed focused on improving RNA yields from FFPE samples. These methods use a combination of optimized lysis buffers and mechanical disruption to break down formalin-induced crosslinks and release RNA more efficiently.

• Proteinase K treatment: This enzyme helps break down protein-RNA crosslinks formed during formalin fixation, allowing for better RNA isolation.
• Heat-induced reversal of crosslinks: The tissues are heated to reverse formalin-induced crosslinking, which improves the release of RNA and increases the yield of usable RNA.

Schematic of formalin-induced crosslink formation and the removal of crosslinked proteins by treatment with proteinase K

These new methods allow for higher-quality RNA extraction from FFPE tissues, improving the material available for sequencing.

2. Optimized Reverse Transcription

Reverse transcription is a key step in RNA sequencing, where RNA is converted into complementary DNA (cDNA) before sequencing. FFPE samples, with their fragmented RNA, present significant challenges at this stage.

Recent Advancements: The use of specialized reverse transcriptase enzymes is one of the main innovations. These enzymes have been engineered to work more efficiently with fragmented RNA from FFPE samples.

• Improved enzyme formulations: New reverse transcriptases have enhanced ability to synthesize cDNA even from highly fragmented RNA. These enzymes typically have better thermal stability and can tolerate RNA degradation, reducing the risk of incomplete or biased cDNA synthesis.
• Modified reaction buffers: These buffers help to reduce the impact of RNA secondary structures and fragmentation, ensuring more accurate cDNA synthesis.

These improvements ensure that even highly degraded RNA from FFPE tissues can be effectively transcribed, enabling more reliable downstream analysis.

3. Enhanced Library Preparation Protocols

Library preparation involves converting RNA (or cDNA) into a sequencing-ready library. This step is essential for enabling high-throughput sequencing technologies to accurately read and map the sequences.

Recent Advancements:

• Fragmentation optimization: FFPE RNA is already fragmented, so some libraries use techniques that take advantage of this natural fragmentation to avoid additional damage. 
• Reduced bias: Protocols designed by companies like Illumina, NEB (New England Biolabs), and others minimize the biases introduced by FFPE degradation and optimize the ligation steps for high-quality library prep. These kits incorporate more efficient adapters and ligation steps that allow for more uniform coverage across transcripts.
• Enrichment of specific RNA species:  Companies have also been incorporating hybridization-based approaches to enrich RNA species (such as mRNA or long non-coding RNA) to reduce sequencing costs and focus on relevant molecular targets.

By optimizing these processes, modern library prep techniques ensure better-quality sequencing data from FFPE tissue samples, despite RNA degradation.

4. Higher Sensitivity Sequencing Technologies

Process: Sequencing technologies have rapidly evolved in terms of sensitivity, allowing researchers to detect even low-abundance transcripts, which is critical when working with degraded RNA.

Recent Advancements:

• Increased read depth: Next-generation sequencing (NGS) platforms now offer higher read depth, which improves the ability to detect low-abundance transcripts. Higher coverage allows for more accurate quantification of gene expression, even when working with small amounts of RNA from FFPE samples.
• Long-read sequencing: Platforms like Pacific Biosciences and Oxford Nanopore have introduced long-read sequencing technologies. These are particularly useful for FFPE samples, where RNA fragmentation can disrupt short-read sequencing methods. Long reads can span across fragmented sequences, improving the overall accuracy of transcript assembly and gene expression profiling.
• Targeted sequencing: This approach focuses sequencing efforts on specific regions of interest (e.g., cancer-related genes). By increasing sequencing depth in specific areas, researchers can obtain high-resolution data without needing to sequence the entire transcriptome, which is especially useful when working with degraded samples.

These advancements in sequencing technologies allow for deeper insights, even when working with challenging FFPE tissue samples.

5. Single-Cell RNA Sequencing Adaptations

Single-cell RNA sequencing (scRNA-seq) is an emerging technology that allows researchers to study gene expression at the individual cell level. When adapted for FFPE tissues, this technique provides a detailed, high-resolution view of cellular heterogeneity, which is particularly important in cancer research.

Recent Advancements:

• Laser capture microdissection (LCM): LCM is used to isolate individual cells or specific regions of interest from FFPE tissue sections. This approach allows for the selective capture of cancerous cells or subpopulations that may have distinct molecular features.
• Drop-seq and other microfluidic methods: New methods that enable the isolation and sequencing of single cells from FFPE samples are rapidly evolving. These microfluidic techniques help prepare single-cell libraries from formalin-fixed tissue, enabling scRNA-seq on a much broader range of FFPE samples.
• Increased sensitivity for low-abundance transcripts: Single-cell RNA sequencing protocols have also been modified to deal with the challenges of low RNA yield from FFPE samples. New amplification techniques have been integrated into the workflows, allowing for better recovery of gene expression profiles from individual cells.

This approach offers unprecedented insights into tumor microenvironments and rare cellular subtypes, even from FFPE samples.

6. Amplification Techniques for Degraded RNA

RNA amplification is necessary when working with FFPE tissues due to the low yield and fragmentation of RNA. This process is essential to ensure enough material is available for sequencing.

Recent Advancements:

• Whole-transcriptome amplification (WTA): WTA techniques have been optimized for FFPE samples to improve RNA yield. This method amplifies RNA from entire transcriptomes, making even small amounts of degraded RNA sufficient for sequencing.
• Polymerase chain reaction (PCR)-based amplification: Recent advances in PCR have improved the efficiency and specificity of amplifying fragmented FFPE RNA. These modifications reduce amplification bias and help preserve the overall gene expression profile.

These amplification techniques are key to increasing the usable RNA from FFPE tissues, enabling more comprehensive transcriptome analysis.

Key Benefits of RNA Sequencing in FFPE Cancer Research

RNA sequencing (RNA-Seq) is a powerful tool for studying gene expression, and its application to FFPE (formalin-fixed paraffin-embedded) cancer tissues offers several key advantages. Here are the main benefits:

1. Enhanced Molecular Profiling

RNA-Seq gives a complete picture of gene expression, helping researchers uncover dysregulated pathways, mutations, and biomarkers in cancer cells. For FFPE tissues—often the only available samples for retrospective studies—it reveals vital molecular characteristics, such as driver mutations, gene fusions, and expression patterns that drive tumor growth.

2. Improved Diagnostic Accuracy

Compared to traditional methods like immunohistochemistry and PCR, RNA-Seq offers a more accurate way to diagnose cancer. By profiling the entire transcriptome, it identifies specific gene expression signatures tied to cancer subtypes, improving tumor classification, detecting rare mutations, and guiding treatment decisions—especially when other methods fall short.

3. Identification of Therapeutic Targets

RNA-Seq allows researchers to pinpoint overexpressed genes and non-coding RNAs that could be targeted by drugs. This is crucial for FFPE samples, where archival tissues can reveal previously unknown targets that are hard to study with older methods, opening doors for new treatment options.

4. Understanding Tumor Heterogeneity

Cancer tumors are diverse, containing various cell types with different gene expression profiles. RNA-Seq captures this heterogeneity, even in FFPE samples, by examining gene expression at a single-cell level or within specific tumor regions. This helps researchers understand tumor behavior, including how different cells contribute to metastasis, drug resistance, and overall cancer progression.

5. Supporting Personalized Medicine

By analyzing the molecular features of an individual’s cancer, RNA-Seq supports personalized medicine. In FFPE samples, it helps researchers track changes over time and identify genetic alterations that may affect treatment response. This makes it easier to match patients with the most effective therapies based on their unique genetic profile.

6. Working with Archived Samples

FFPE samples are often the only source for historical cancer data, especially when fresh biopsies aren’t possible. RNA-Seq extracts valuable data from these archived samples, enabling comparisons with modern cancer datasets and tracking changes in tumor biology over time.

7. Exploring Non-Coding RNAs

Beyond protein-coding genes, RNA-Seq also looks at non-coding RNAs like microRNAs and long non-coding RNAs, which play critical roles in cancer. Even in degraded FFPE samples, these molecules provide important insights into cancer development, metastasis, and resistance to treatment.

Hence, RNA sequencing of FFPE cancer tissues brings invaluable benefits to cancer research, providing a comprehensive, accurate, and detailed view of gene expression. 

Challenges in RNA Sequencing with FFPE Samples

RNA sequencing from FFPE samples presents several challenges due to the preservation methods involved.

• RNA Degradation: Formalin fixation causes crosslinking between proteins and RNA, leading to degradation. The process results in fragmented RNA, making it difficult to obtain high-quality sequencing data.
• Low RNA Yield: Due to the preservation process, the RNA yield from FFPE samples is often low, which can limit the amount of usable material for sequencing.
• Chemical Modifications: Formalin-induced modifications, such as base modifications and strand breaks, can interfere with RNA extraction and sequencing efficiency.
• Bias in Gene Expression Profiling: RNA degradation and chemical modifications can lead to biased gene expression profiles, making it harder to interpret the data accurately.
• Challenges in Library Preparation: FFPE RNA requires optimized protocols for library preparation to improve the quality of sequencing results. Existing commercial kits often need adjustments when working with FFPE tissue, adding complexity to the workflow.
• Difficulty in Analyzing Low-Abundance Transcripts: Low-abundance transcripts may be underrepresented due to the degradation of RNA, making it challenging to detect less-expressed genes or variants.

These challenges make RNA sequencing from FFPE samples more complex.

To Sum Up!

RNA sequencing has revolutionized cancer research, especially for FFPE tissues, which were once difficult to analyze due to RNA degradation. Recent advancements in RNA extraction, reverse transcription, and sequencing technologies now allow researchers to extract valuable data from these archived samples, aiding in more precise cancer diagnoses, identifying therapeutic targets, and exploring tumor heterogeneity.

Using RNA sequencing for FFPE samples reveals molecular details hidden in degraded tissues. With continuous advancements in sequencing technology, these methods are pushing the boundaries of cancer research and treatment.

At Biostate AI, we offer affordable, high-quality RNA sequencing services, including FFPE samples. Our advanced multiomics solutions provide deep insights into cancer research while keeping costs low. We integrate RNA, DNA, and methylation data for a comprehensive approach to molecular cancer studies.

Starting at just $80 per sample, Biostate AI ensures accurate, actionable results for any sample type, including FFPE tissues and blood. Whether you’re tracking longitudinal changes or gene expression patterns, we make sure you get the insights you need with minimal effort and cost.

Ready to take your cancer research to the next level? Get a quote for your experiments and learn how Biostate AI can support your work with high-quality, cost-effective RNA sequencing solutions.