EZ1&2 DNA FFPE Kits

For automated purification of gDNA from FFPE tissues using the EZ1 Advanced XL or EZ2 Connect instrument

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EZ1&2 DNA FFPE Kit (48)

Cat. No. / ID: 954404

For 48 preps: EZ1&2 DNA FFPE cartridge, disposable filter-tips and tip-holders, tubes, Paraffin Removal Solution, Proteinase K, RNase A, RNase-free water and buffers

KitKit componentAccessories

DNA FFPE

DNA FFPE UNG

Uracil-N-Glycosylase

EZ1 Advanced XL DNA FFPE Card

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EZ1&2 DNA FFPE Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

High recovery of amplifiable DNA
Paraffin removal without xylene or similar solvents
Option to remove uracil (deaminated cytosine) artifacts
Ready-to-use DNA for PCR, digital PCR (dPCR) and next-generation sequencing (NGS)
Automated heating and reagent-pipetting pretreatment steps, available on the EZ2 Connect

Product Details

Purify large amounts of amplifiable DNA from hard-to-lyse formalin-fixed, paraffin-embedded (FFPE) tissue. The EZ1&2 DNA FFPE protocol uses double lysis to recover DNA effectively, while the optional uracil-N-glycosylase (UNG) step removes deaminated cytosine artifacts to limit the risk of nucleotide read errors.

The EZ1&2 DNA FFPE protocols are automatable on either the EZ1 Advanced XL or the EZ2 Connect.

The EZ2 Connect features cardless protocols, expanded applications and remote connectivity options. Click here to learn more about the EZ2 Connect, or contact your sales representative to discover how easily you can get an EZ2 Connect for your lab.

Performance

Under UV-vis, fluorometric, and qPCR analysis, most of the samples processed with the EZ1&2 DNA FFPE Kit showed a higher abundance of dsDNA (see figure “ Optimized for PCR performance”).

For NGS analysis, the EZ1&2 DNA FFPE UNG Kit significantly reduces artificial C→T/G→A transitions, which commonly occur in FFPE material due to cytosine deamination (see “ Artificial C→T/G→A transitions”). This helps prevent false-positive reports of single-nucleotide variants (SNVs) in NGS (see figure “ Reduction in artifactual C→T | G→A transitions”).

See figures

Optimized for PCR performance.

Artificial C→T/G→A transitions.

Reduction in artifactual C→T | G→A transitions.

Principle

The EZ1&2 DNA FFPE Kit maximizes DNA yields from limited sample inputs in two ways: By implementing a two-step lysis procedure, it helps ensure high DNA extraction even from difficult-to-lyse samples. In addition, its wash-free no-solvent deparaffinization technique minimizes the risk of losing of any sample material, which is difficult to avoid with multiple-wash solvent-based deparaffinization methods.

Crosslink removal further increases the amount of recovered amplifiable DNA (see figure “ Optimized for PCR performance”). In addition, it also makes the recovered DNA especially suitable for NGS analysis (see figure “ Reliable dPCR and NGS results”).

See figures

Optimized for PCR performance.

Reliable dPCR and NGS results.

Procedure

The EZ1&2 DNA FFPE Kit fully automates the bind, wash and elute steps on the EZ1 Advanced XL or the EZ2 Connect. The preparation steps (i.e., Proteinase K digestions, crosslink removal and RNase A digestion) are done manually on the EZ1 Advanced XL; on the EZ2 Connect, many of these preparation steps can be automated (see figure “ EZ1&2 DNA FFPE workflow”).

See figures

EZ1&2 DNA FFPE Kit workflow.

Applications

DNA isolated with the EZ1&2 DNA FFPE Kit can be used immediately for PCR, dPCR or NGS. Alternatively, it can be stored at −30 to −15°C.

Supporting data and figures

Artificial C→T/G→A transitions.

Sample fixation can lead to artifacts in the DNA sequence. The most common artifact in FFPE tissues is the deamination of cytosine to uracil, which results in a single nucleotide variation (SNV).

Specifications

Features	Specifications
Applications	PCR, digital PCR, next-generation sequencing
Elution volume	60 and 100 µl
Format	Magnetic bead
Main sample type	Formalin-fixed paraffin-embedded tissue samples
Processing	Automated with the EZ1 Advance XL or EZ2 Connect
Purification of total RNA, miRNA, poly A+ mRNA, DNA or protein	Genomic DNA
Technology	Silica technology
Sample amount	Tissue sections, each with a thickness of 5–10 µm, for a total volume of 4 mm3

Resources

Eva Haenssler, Simon Hertlein, Christina Fischer, Nicole Pickave, Martin Schlumpberger
QIAGEN Strasse 1, 40724 Hilden, Germany

FAQ

If it is more convenient, either the first or the second Proteinase K lysis step in the EZ1&2 DNA FFPE Kits workflow can be performed overnight. This will not affect the DNA quality and will also not increase yields.

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Buffer FTB provides optimized lysis conditions that additionally allow the specific removal of deaminated cytosine residues by the enzyme Uracil-N-Glycosylase (UNG) in the EZ1&2 DNA FFPE UNG workflow.

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DNA from FFPE samples is often fragmented, yielding DNA with a broad size distribution depending on multiple factors such as fixation and storage conditions. The EZ1&2 DNA FFPE Kits provide an optimized workflow for extraction of DNA for use in short amplicon PCR, dPCR, and next-generation sequencing analysis using targeted DNA panels.

In case FFPE samples are of high quality and allow the extraction of more intact DNA, we offer a supplementary protocol for downstream applications that require larger DNA fragments. These applications include for instance large amplicon PCR (>0.5 kb) and long-read DNA sequencing.

High DNA integrity can be presumed if formalin fixation was less than 24 hours and the sample has been stored at low temperature (4–8°C or −20°C) or for a short period of time (e.g., up to a few weeks). For these samples, the supplementary protocol Extraction of more intact DNA from FFPE tissue material using the EZ1&2 DNA FFPE Kits with Buffer LF is suitable as it protects and best preserves the DNA size distribution present in the original FFPE sample during DNA extraction.

Please contact Technical Service to inquire about the supplementary protocol and the availability of Buffer LF.

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The 2nd Proteinase K step improves lysis efficiency and yields, in particular, for difficult-to-lyse tissue material. Hence, omission of this step may result in decreased yields.

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This can vary significantly and strongly depends on the DNA quality present in the original FFPE sample. Formalin fixation, paraffin embedding, and storage conditions are factors that affect the DNA size distribution and may cause significant fragmentation of nucleic acids. To limit the extent of nucleic acid fragmentation, be sure to:

Fix tissue samples in 4%–10% formalin as quickly as possible after surgical removal.
Use a fixation time of 14–24 hours (longer fixation leads to more fragmentation, reducing performance in downstream assays).
Thoroughly dehydrate samples prior to embedding (residual formalin can inhibit Proteinase K digestion).

Store FFPE tissue samples at 4–8°C.

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QIAGEN’s Deparaffinization Solution (DPS) (cat. no. 939018) can be used for removal of paraffin. For this, adhere to the following procedure:

Place the FFPE sections in a 1.5 ml or 2 ml microcentrifuge tube (not supplied). Add 300 μl DPS, vortex vigorously for 10 seconds, and centrifuge briefly to bring the sample to the bottom of the tube.
Incubate at 56°C for 3 minutes, and then allow to cool to room temperature.

Note: If too little DPS is used or if too much paraffin is carried over with the sample, DPS may become waxy or solid after cooling. If this occurs, add additional DPS and repeat the incubation at 56°C. After this, proceed with step 3 of the Quick-Start Protocol for the EZ1&2 DNA FFPE and EZ1&2 DNA FFPE UNG Kits.

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After 90°C incubation for cross-link removal, sample lysates can be stored at 4–8°C for up to one week and at –20°C or –80°C for up to 4 weeks. The upper phase of Paraffin Removal solution should be removed before storage. Before proceeding with the workflow after storage, thaw frozen samples at room temperature for 30 minutes or allow refrigerated (4–8°C) samples to equilibrate to room temperature for 15–30 minutes.

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A temperature range of 56–68°C works fine for the second Proteinase K lysis step. However, we recommend following the instructions in handbook and perform the second lysis step at 65°C.

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