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EZ1&2 DNA FFPE Kits

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

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
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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
$530.00
KitKit component
DNA FFPE
DNA FFPE UNG
Uracil-N-Glycosylase
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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

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

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

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 EZ2 Advanced XL; on the EZ2 Connect, many of these preparation steps can be automated (see figure “ EZ1&2 DNA FFPE workflow”).

See figures

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

Specifications

FeaturesSpecifications
ApplicationsPCR, digital PCR, next-generation sequencing
Elution volume60 and 100 µl
FormatMagnetic bead
Main sample typeFormalin-fixed paraffin-embedded tissue samples
ProcessingAutomated with the EZ1 Advance XL or EZ2 Connect
Purification of total RNA, miRNA, poly A+ mRNA, DNA or proteinGenomic DNA
Sample amountTissue sections, each with a thickness of 5–10 µm, for a total volume of 4 mm3
TechnologySilica technology

Resources

Kit Handbooks (1)
Safety Data Sheets (2)
Download Safety Data Sheets for QIAGEN product components.
Download Safety Data Sheets for QIAGEN product components.

FAQ

Can samples be lysed overnight with Proteinase K in the EZ1&2 DNA FFPE procedure?

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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What is the difference between Buffer FTB and Buffer ATL?

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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Are there important considerations for plasma generation and urine handling?

It is strongly advised to follow the recommendations for preparing sample material provided in the corresponding Protocol Sheet to ensure reliable results.

Plasma: It is recommended to perform plasma separation immediately after blood collection when using EDTA or citrate as anticoagulant to prevent the release of genomic DNA into the plasma fraction.

Urine: Because circulating cell-free DNA in non-stabilized urine samples is rapidly degraded after sample collection due to high nuclease activity, eluates may contain no DNA or exhibit low DNA concentration. Therefore, it is recommended to stabilize urine samples. Even when using stabilized urine, it is recommended to perform a centrifugation step immediately after stabilization to prevent the release of genomic DNA from cells. Alternatively, non-stabilized urine samples can be processed immediately after collection and centrifugation using ATL-pretreatment and automated DNA extraction as described in the corresponding Protocol Sheet.

FAQ ID - 3699
Is the DNA extracted with the EZ1&2 DNA FFPE Kits suitable for downstream applications that require more intact DNA such as long-range PCR (>1 kb) and long-read DNA sequencing?

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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Can the 2nd Proteinase K step be omitted?

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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What size of DNA can be expected?

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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Are there other options for paraffin removal?

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

  1. 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.
  2. 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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What are recommended stopping points in the procedure of the EZ1&2 DNA FFPE Kits?
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 (48°C) samples to equilibrate to room temperature for 1530 minutes.
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Can the second Proteinase K lysis step in the EZ1&2 DNA FFPE procedure be carried out at lower temperatures than 65°C?

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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