FFPE High-Throughput Sample Prep: Protein Extraction and Nucleic Acid Shearing

With the high-throughput ultrasonicator UIP400MTP, Hielscher Ultrasonics addresses the challenges of formalin fixation and paraffin embedded (FFPE) tissue preparation. Learn how ultrasonication processes FFPE sample in large numbers for FFPE deparaffinization, tissue lysis, homogenization, protein extraction and DNA/RNA shearing! Take advantage of ultrasonic FFPE tissue preparation – processing large sample numbers in multiwell plates! Obtain high-quality samples and get high sample numbers at for reliable research results! And last, but not least save time and money!

FFPE Sample Preparation Facilitated by High-Throughput Sonication

Formalin fixation and paraffin embedding (FFPE) is the most common method for preserving and archiving solid tissues. The extraction of biomolecules from FFPE tissue samples often presents significant challenges due to the quality of the stored samples. These samples, which are invaluable assets in molecular biology and clinical research, provide a rich source of biological information for retrospective studies and diagnostic biomarker validation. However, the process of formalin fixation and paraffin embedding, while preserving tissue architecture and morphology, complicates the extraction of high-quality nucleic acids and proteins. Formalin induces cross-linking of nucleic acids and proteins, leading to molecular fragmentation and chemical modifications. Learn how the high-throughput ultrasonicator UIP400MTP overcomes the challenges of FFPE sample preparation!

Ultrasonicator for Efficient FFPE Sample Preparation

  • Easy-to-Use Workflow: Simplified processes that are user-friendly.
  • Deparafinization, Protein Extraction, DNA / RNA Shearing
  • Rapid High-Throughput Processing: Efficient handling of multi-well plates.
  • Effective Deparaffinization: Improved solubilization of proteins.
  • Non-Toxic Solvents: Avoids the use of harmful organic solvents such as xylene.

 

The UIP400MTP multiwell plate ultrasonicator can process FFPE samples in high-throughput for protein extraction and DNA and RNA shearing

UIP400MTP high-throughput sonicator for high-throughput FFPE sample processing in multiwell plates

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FFPE Sample Preparation using Ultrasonication - UIP400MTP multwell plate sonicator by Hielscher
 

Advancements in Protein Extraction Techniques from FFPE Tissue

Hielscher Ultrasonics addresses the challenges in high-throughput FFPE sample preparation. Sonication employs ultrasonic waves to generate mechanical vibrations and focused cavitation, effectively disrupting cellular structures and enhancing the solubilization of biomolecules. This technique has gained popularity for its ability to increase the efficiency and yield of nucleic acid and protein extraction from FFPE tissues as well as DNA and RNA shearing for library preparation. It is very important to highlight that ultrasonication using the UIP400MTP multiwell plate sonicator maintains the integrity of these biomolecules for downstream applications.

 

The video shows the ultrasonic sample preparation system UIP400MTP, which allows for the reliable sample preparation of any standard multi-well plates using high-intensity ultrasound. Typical applications of the UIP400MTP include cell lysis, DNA, RNA, and chromatin shearing as well as protein extraction.

Ultrasonicator UIP400MTP for multi-well plate sonication

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Nucleic Acid Shearing using High-Throughput Ultrasonication

96-well plates and other multiwell plates are best processed using the sonicator UIP400MTP. This ultrasonic system is ideal for lysis, DNA fragmentation and cell solubilization processing samples in high-throughput.The multi-well ultrasonicator UIP400MTP for the use in high-throughput settings brings the preparation of FFPE samples to new level. This multiwell-plate sonication method provides an efficient and reliable solution for the simultaneous processing of multiple samples. It facilitates the rapid and reproducible extraction of DNA, RNA, and proteins, which are critical for various analytical techniques, including next-generation sequencing (NGS), quantitative PCR, and proteomic analyses. Optimization of sonication parameters, such as amplitude, duration, and temperature, further enhances the quality and quantity of the extracted biomolecules.

The UIP400MTP sonicator for multiwell plates offers significant advantages for the fragmentation and shearing of DNA and RNA from FFPE tissue. One of the standout features of this system is its ability to achieve narrow fragment sizes of DNA and RNA, providing precise tunability of sonication intensity to obtain short fragments of 150-200 base pairs (bp) or longer fragments of 15-20 kilobase pairs (kbp). This versatility makes the UIP400MTP indispensable for both short-read and long-read sequencing applications, ensuring high-quality results for next-generation sequencing (NGS) and whole-genome sequencing (WGS). Its precise control over fragment size is crucial for researchers in all fields of genomics, as it allows to prepare samples according to specifications.

Contact Us for Advanced Solutions in FFPE Tissue

Discover the UIP400MTP multiwell plate sonicator for efficient biomolecule recovery from FFPE samples, maintaining the integrity of extracted nucleic acids and proteins and ensuring reproducibility of results. This technology integrates seamlessly with other preparative and analytical workflows, streamlining and enhancing molecular investigations using FFPE tissue archives.

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Fixatives and Their Effects

Fixation is a crucial step in sample preparation that preserves cellular structures, halts biochemical reactions, and prevents degradation. Various fixatives are employed depending on the specific experimental requirements. The two most common fixatives are formaldehyde and paraformaldehyde, which crosslink proteins and nucleic acids, preserving the morphology and antigenicity of cells and tissues. Other fixatives, such as ethanol, methanol, and glutaraldehyde, are used for specific applications.

Formaldehyde and paraformaldehyde fixatives form methylene bridges between amino groups, resulting in protein crosslinking. This process effectively immobilizes cellular components, preserving their integrity during subsequent analysis steps. The effects of these fixatives can be influenced by factors such as concentration, pH, and temperature, and optimizing these parameters is crucial to ensure optimal preservation of cellular structures.

 
Advantages of Ultrasonic FFPE Preparation
 

Ultrasonication is a powerful technique for disrupting fixated cells and tissues that excels conventional techniques. It offers several notable advantages over traditional lysis methods:

  • Speed and Efficiency: Ultrasonic lysis provides rapid disruption of cells and tissues, significantly reducing processing time compared to mechanical or chemical lysis methods. High-frequency sound waves generated by the ultrasonic probe create mechanical shear forces, causing disruption of the fixated cellular structures. This fast and efficient disruption enables researchers to process large sample volumes within a short timeframe.
  • Gentle and Adjustable: Ultrasonic lysis offers a gentle disruption mechanism that minimizes damage to sensitive biomolecules such as proteins, nucleic acids, and enzymes. Unlike mechanical methods that generate excessive heat or shear forces, ultrasonic lysis utilizes controlled cavitation to disrupt cells while preserving the integrity and functionality of the intracellular components.
  • Versatility: Ultrasonic lysis can be applied to various fixatives, allowing researchers to work with a wide range of fixated samples. Whether using formaldehyde, paraformaldehyde, or alternative fixatives, ultrasonic lysis consistently delivers efficient disruption, ensuring optimal recovery of cellular components.
  • High Yield and Quality: Ultrasonic lysis facilitates high yields of intact cellular components due to its ability to disrupt fixated cells and tissues uniformly. This enables downstream applications such as protein analysis, nucleic acid extraction, and enzymatic assays to yield reliable and reproducible results.
  • Automation Compatibility: Ultrasonic lysis can be easily integrated into automated systems, allowing for high-throughput sample processing. This compatibility enables researchers to streamline their workflow and increase productivity, particularly in large-scale studies.
96-Well Plate Sonicator UIP400MTP for cell lysis, DNA extraction, DNA fragmentation, cell solubilization and protein purification.

96-well plate sonicator UIP400MTP for the sonication of microtiter and multiwell plates

Ultrasonic lysis has revolutionized the disruption of fixated cells and tissues, offering numerous advantages over traditional lysis methods. Its speed, efficiency, selectivity, versatility, high yield, and automation compatibility make it an indispensable tool in molecular biology and biotechnology research. Offering non-contact sonicators as well as probe-type sonicator, Hielscher Ultrasonics offers the most suitable ultrasonic homogenizer for your life science application. Whether you want to process single samples, multiple sample or very high sample numbers simultaneously, we will offer you the best sonicator matching your research and diagnostic requirements.
Read more about Hielscher non-contact sonicators for multi-sample and high-throughput sample preparation!

FFPE Sample Prep with the UIP400MTP Multiwell-Plate Sonicator

  • one-time investment
  • use your own consumables
  • no reoccurring costs for proprietary accessories and consumables
  • high-throughput
  • precision control
  • state-of-the-art technology
  • reliability & robustness
  • adjustable, precise process control
  • industrial grade: can be continuously operated 24/7
  • easy and safe to operate
  • low maintenance

 
Read more about the applications of sonicators in life science!

Sonicator for high-throughput sample preparation! The UIP400MTP plate sonicator facilitates lysis, protein extraction, DNA fragmentation and cell solubilization of biological samples in 96-well plates.

Plate sonicator UIP400MTP for any 96-well plates, microtiter plates and multi-well plates.

Design, Manufacturing and Consulting – Quality Made in Germany

Hielscher ultrasonicators are well-known for their highest quality and design standards. Robustness and easy operation allow the smooth integration of our ultrasonicators into industrial facilities. Rough conditions and demanding environments are easily handled by Hielscher ultrasonicators.

Hielscher Ultrasonics is an ISO certified company and put special emphasis on high-performance ultrasonicators featuring state-of-the-art technology and user-friendliness. Of course, Hielscher ultrasonicators are CE compliant and meet the requirements of UL, CSA and RoHs.

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High-Throughput RNA-Sequencing in Multi-Well-Plate

UIP400MTP Ultrasonicator: High-Throughput FFPE sample prep in Multi-Well-Plate



FAQ
Below, we answer frequently asked questions, which are relevant to FFPE tissue preparation and ultrasonication of FFPE samples.

How is FFPE tissue prepared?

Steps for FFPE Tissue Preparation: The meticulous handling and processing of fresh tissue are critical for generating high-quality FFPE samples. Ensuring the preservation of cellular architecture, nucleic acids, and proteins is essential for accurate downstream analysis. Each step — from collection to embedding — requires precision to maintain the integrity of the sample for various analyses, including histological examination, immunohistochemistry, and molecular studies. Properly executed, this fixation and embedding process ensures that the preserved tissue accurately reflects the in vivo state, enabling reliable diagnostic and research outcomes.
We walk you through the 6 major steps of the embedding process of FFPE tissue samples.

  • Tissue Collection
    Biopsies from live mammals and tissue cultures are both viable sources for obtaining fresh tissue for FFPE sample preparation.
    It is important to use an aseptic technique: Use sterile instruments and gloves to avoid contamination. Ideally, collect tissues in a sterile environment, such as a surgical suite or laminar flow hood.
    As the specimen is very fragile, its sensitive handling is essential: Minimize delays in processing and immediately start subsequent the tissue processing after the excision. This is crucial to prevent autolysis and degradation. Keep the tissue at room temperature; avoid freezing as it can cause ice crystal formation and tissue damage.
  • Tissue Fixation
    First, the tissue is treated with a fixative solution: Use 10% neutral-buffered formalin (NBF), which is equivalent to 4% formaldehyde in water, buffered to a neutral pH.
    Submerge the tissue completely in formalin. Ensure a fixative-to-tissue volume ratio of at least 10:1. Fixation time typically ranges from 6 to 24 hours, depending on tissue type and size. It is important that the fixative can penetrate the tissue thoroughly. However, over-fixation can lead to cross-linking that complicates antigen retrieval, while under-fixation can result in poor tissue preservation.
  • Tissue Trimming
    Secondly, trim the tissue to a thickness of about 3-5 mm to allow adequate penetration of fixative. Ensure proper orientation of the tissue to capture relevant histological structures. This facilitates the extraction process when the tissue is later used for analysis.
  • Processing the Fixated Sample
    Now, the fixed tissue must be dehydrated: After fixation, the tissue needs to be dehydrated to ensure thorough penetration by the paraffin wax. Pass the tissue through a graded series of ethanol (70%, 80%, 90%, and 100%) to remove water.
    Clearing with xylene: Paraffin wax is insoluble in water, but soluble in xylene. Therefore, the water in the tissue must be replaced with xylene. However, xylene itself is insoluble in water but soluble in alcohol, necessitating an intermediate step where water is first replaced with alcohol.Immerse the tissue in xylene or a xylene substitute to remove ethanol and prepare the tissue for paraffin infiltration.
    Infiltration using paraffin: Embed the tissue in molten paraffin wax, ensuring complete infiltration. This step typically involves several changes of paraffin to ensure thorough impregnation.
  • Embedding the Tissue
    In this step, the tissue is molded into a tissue block: Place the tissue in a mold with the desired orientation and pour molten paraffin over it. Allow the paraffin to solidify by cooling at room temperature or on a cold plate.
  • Sectioning and Mounting
    Microtomy: To slice the embedded tissue, use a microtome to cut thin sections (typically 4-5 micrometers) from the paraffin block. Then the sample is mounted, placing the sections on glass slides for subsequent staining and microscopic analysis.
    Finally, check the quality of the histology: Evaluate the first sections under a microscope to ensure proper fixation and processing. Adjust protocols as needed based on tissue type and observed quality.

 
FFPE tissues can be used to recover RNA, DNA, and proteins as well as discover signs of cancer or other disease. They can be stored for years, and are an essential part of how researchers and doctors leverage tissue samples for diagnosis and research.

What are common problems and challenges with FFPE tissue?

Formalin-Fixed, Paraffin-Embedded (FFPE) tissue samples are widely used in research and diagnostics, but they present several challenges and common problems:

  • Degradation of Biomolecules: Prolonged fixation can lead to the degradation of DNA, RNA, and proteins, making it difficult to extract high-quality nucleic acids or proteins for downstream applications. Correct fixation (avoiding under- and over-fixation) is essential for tissue preservation.
  • Cross-linking: Formalin fixation causes cross-linking of proteins and nucleic acids, which can hinder molecular analysis and affect the accuracy of immunohistochemistry and other assays.

  • Antigen Masking: The fixation process can mask antigenic sites, reducing the effectiveness of antibody binding in immunohistochemistry and other immunological assays. This often requires antigen retrieval, a procedures through that the masking of an epitope is reversed and epitope-antibody binding is restored. However, full antigenicity may not always be restored.
  • Variable Fixation Quality: Differences in fixation times and conditions can lead to inconsistent sample quality, affecting reproducibility and comparability of results. Use reliable fixation protocols and avoid under- and over-fixation.
  • DNA Damage and Fragmentation: Formalin fixation of FFPE samples can cause various types of DNA damage, including cytosine deamination (C to T mutations), oxidative damage (e.g., 8-oxo-guanine leading to G to T mutations), as well as physical disruptions such as nicks, gaps, and abasic sites that hinder DNA polymerase activity. The formalin fixation process can cause fragmentation of DNA, complicating genetic and genomic analyses such as PCR and sequencing.
  • RNA Quality: RNA extracted from FFPE tissues is often fragmented and chemically modified, making it challenging to perform high-quality transcriptomic analyses.
  • Protein Modifications: Formalin can induce chemical modifications in proteins, affecting their structure and function, which can interfere with proteomic analyses.
  • Sample Processing Artifacts: During the embedding and sectioning process, mechanical stress and heat can introduce artifacts and cause further damage to the tissue.
  • Batch-to-Batch Variability: Variations in fixation and embedding protocols between different batches can lead to significant variability in the results, complicating comparisons between studies.
  • Storage Issues: Long-term storage of FFPE blocks can lead to additional degradation and loss of nucleic acid integrity over time, impacting the viability of archival samples for retrospective studies.

Using optimized protocols, careful sample handling, and applying advanced techniques help to improve the quality and reliability of data obtained from FFPE tissues.

What is the difference between FFPE and frozen tissue?

FFPE (Formalin-Fixed, Paraffin-Embedded) tissue is preserved using formalin to fix the tissue and then embedded in paraffin wax, allowing for long-term storage at room temperature while maintaining tissue morphology. In contrast, frozen tissue is rapidly preserved by freezing, which better maintains the integrity of nucleic acids and proteins but requires storage at very low temperatures.

What chemicals are used for FFPE embedding?

The chemicals used for FFPE embedding normally include formalin for fixation and paraffin wax for embedding. For FFPE embedding, tissues are typically fixed using either 10% (v/v) neutral buffered formalin (FA) or a freshly prepared 4% (w/v) formaldehyde solution (PFA) made from paraformaldehyde powder. Formalin, which is a solution of formaldehyde in water, is buffered to a neutral pH to preserve tissue morphology and prevent excessive cross-linking. The paraformaldehyde-based solution also provides effective fixation by cross-linking proteins, thereby stabilizing the tissue structure for subsequent embedding in paraffin wax. These chemicals are essential for maintaining tissue integrity and morphology during the fixation and embedding process.

How is the paraffin remove from FFPE samples?

To remove paraffin from FFPE samples, the tissue sections are typically subjected to a series of xylene washes followed by rehydration through a graded series of alcohols and finally water. Since xylene is highly toxic, posing health risks such as respiratory issues, skin irritation, and potential long-term effects with repeated exposure, ultrasonic paraffin removal is emerging as a promising alternative in many laboratories. This method uses intense ultrasonic waves to efficiently and safely remove paraffin without the need for toxic solvents like xylene, thereby reducing the risk to laboratory personnel and creating a safer working environment.

How long should I fixate my tissues for good FFPE sample quality?

General recommendations for fixation duration typically suggest fixing tissue samples in formalin for 24 to 48 hours. This duration is generally sufficient to preserve tissue morphology and cellular structures while minimizing over-fixation, which can lead to excessive cross-linking and degradation of nucleic acids and proteins. However, the optimal fixation time can vary depending on the size and type of tissue, with smaller or more delicate samples requiring shorter fixation times. It is crucial to balance adequate fixation to prevent tissue autolysis and degradation while avoiding prolonged fixation that could complicate downstream molecular analyses.

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