QIAamp DNA FFPE Advanced Kit – FFPE Tissue DNA Extraction

Next-generation DNA isolation for formalin-fixed paraffin-embedded (FFPE) tissues

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QIAamp DNA FFPE Advanced Kit (50)

Cat. No. / ID: 56604

For 50 preps: QIAamp UCP MinElute Columns, collection tubes, Deparaffinization Solution, Proteinase K, RNase A, RNase-free water and buffers

ProductKit component

QIAamp DNA FFPE Advanced Kit (50)

QIAamp DNA FFPE Advanced UNG Kit (50)

Uracil-N-Glycosylase

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QIAamp DNA FFPE Advanced 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
Uracil (deaminated cytosine)–artifact removal step using uracil-N-glycosylase (UNG) during DNA extraction
Ready-to-use DNA for PCR, digital PCR (dPCR) and next-generation sequencing (NGS)

Product Details

Increase your recovery of high-quality DNA from FFPE tissue with the QIAamp DNA FFPE Advanced Kits’ xylene-free, no-wash deparaffinization, double-lyse protocol, and UCP (ultra-clean production) spin column technology.

Remove C→U transitions of nucleic acids with the kits’ optional UNG digest to optimize your recovered DNA for NGS analysis.

You can also automate the QIAamp DNA FFPE Advanced protocols on the QIAcube Connect.>

Performance

One characteristic of FFPE DNA quantification is that different methods will give out different results, and high UV-vis or fluorometric values do not necessarily mean good PCR performance.

Because real-time PCR, or quantitative PCR (qPCR), is one of the most common downstream applications for FFPE, the QIAamp DNA FFPE Advanced Kits are optimized for qPCR.

Regardless of values obtained in UV-vis or fluorometric measurements, QIAamp DNA FFPE Advanced Kits consistently showed better PCR performance under qPCR quantification than the other products tested (see figure “ Optimized for PCR performance”).

The QIAamp DNA FFPE Advanced UNG Kit is also well suited for purifying DNA to be used in NGS analysis, because it addresses the issue of artificial C→T/G→A transitions, which commonly occur in FFPE material due to cytosine deamination (see “ Artificial C→T/G→A transitions”).

Through UNG-based uracil digestion during DNA isolation, the QIAamp DNA FFPE Advanced UNG protocol reduces false-positive reports of single-nucleotide variants (SNVs) in NGS (see figure “ Dramatic reduction in artifactual C→T | G→A transitions”).

See figures

Optimized for PCR performance.

Artificial C→T/G→A transitions.

Dramatic reduction in artifactual C→T | G→A transitions.

Principle

There are three major challenges in preparing DNA from FFPE tissues:

Low yields due to limited input material and compromised DNA
Nonamplifiable DNA due to formalin-induced crosslinks
Deaminated-cytosine artifacts can cause false results in NGS for mutational analyses

The QIAamp DNA FFPE Advanced Kits maximize DNA yields from limited sample inputs in two ways:

By implementing a two-step lysis procedure, which ensures high DNA extraction even from difficult-to-lyse samples
By replacing solvent-based paraffin removal with the use of Deparaffinization Solution, which eliminates all wash steps before initial lysis, thus minimizing the risk of losing scarce sample material

Crosslink removal further increases the recovery of amplifiable DNA (see figure “ Optimized for PCR performance”).

Optional UNG treatment before the second lysis removes deaminated-cytosine artifacts, making the DNA especially suitable for NGS analysis (see table “ Primed for NGS”, as well as figures “ Reliable dPCR and NGS results” and “ Dramatic reduction in artifactual C→T | G→A transitions”).

See figures

Optimized for PCR performance.

Primed for NGS.

Reliable dPCR and NGS results.

Dramatic reduction in artifactual C→T | G→A transitions.

Procedure

The QIAamp DNA FFPE Advanced procedure consists of a simplified deparaffinization step, two lysis steps with in-between de-crosslinking and optional artifact removal, and the standard bind-wash-elute steps (see figure “ QIAamp DNA FFPE Advanced workflow”).

See figures

QIAamp DNA FFPE Advanced workflow.

Applications

DNA from FFPE using the QIAamp DNA FFPE Advanced Kits can be used immediately for PCR, dPCR or NGS, or it can be stored at −30 to −15°C.

Supporting data and figures

QIAamp DNA FFPE Advanced workflow.

The QIAamp DNA FFPE Advanced procedure removes paraffin without the tediousness of multiple wash steps, by using Deparaffinization Solution instead of xylene or any other solvent.

Lysis is done with Proteinase K. Cross-links are removed by heat incubation at 90°C for 1 hour. Artifacts can be removed with UNG.

RNA is then digested, and the sample is lysed a second time to increase DNA recovery.

DNA is bound with the QIAamp UCP MinElute spin column. Contaminants are washed off using Buffer AW1, Buffer AW2 and ethanol, and DNA is eluted in Buffer ATE.

Specifications

Features	Specifications
Applications	PCR, digital PCR, next-generation sequencing
Elution volume	20–100 µl
Format	Spin column
Main sample type	Formalin-fixed paraffin-embedded (FFPE) tissue samples
Processing	Manual or automated with the QIAcube Connect
Purification of total RNA, miRNA, poly A+ mRNA, DNA or protein	Genomic DNA
Sample amount	Tissue sections, each with a thickness of 5–10 µm, for a total volume of 4 mm³
Technology	Silica technology

Resources

Sample to Insight solutions for successful molecular analysis

Critical factors for molecular analysis of FFPE samples

For QIAamp DNA FFPE Advanced Kit (cat. no. 56604)

For QIAamp DNA FFPE Advanced UNG Kit (cat. no. 56704)

FAQ

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 QIAamp DNA FFPE Advanced 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 FFFPE 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 (>0,5kb) 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 QIAamp DNA FFPE Advanced 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 TechService to inquire about the supplementary protocol and the availability of Buffer LF.

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A temperature range of 56°C - 68°C works fine for the second Proteinase K lysis step. However, we recommend following the instructions in the QIAamp DNA FFPE Advanced Handbooks and perform the second lysis step at 65°C.

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Buffer FTB provides optimized lysis conditions, and additionally allows the specific removal of deaminated cytosine residues by the enzyme Uracil-N-Glycosylase (UNG) in the QIAamp DNA FFPE Advanced UNG workflow.

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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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Size distribution of the extracted DNA can vary significantly and first and foremost 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 h (longer fixation times lead to more severe DNA fragmentation, resulting in poor 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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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 blue phase of Deparaffinization 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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If it is more convenient either the first or the second Proteinase K lysis step in the QIAamp DNA FFPE Advanced workflow can be performed overnight. This will not affect the DNA quality but also not increase yields.

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