Qiagen, 19160, Uracil-N-Glycosylase (2 x 1 ml)

50 preps: For use with QIAGEN DNA FFPE UNG kits

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 ”).

Principle

- 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

- 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

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

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

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 ”).

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 ”).

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.