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PAXgene Tissue DNA Kit

For isolation and purification of DNA from tissues fixed and stabilized using the PAXgene Tissue System

  • Effective purification of high-quality, unmodified DNA from tissue with preserved morphology
  • Protocols for paraffin-embedded or paraffin-free tissues
  • Automated purification on the QIAcube

Feature

Specification

Format

Spin column

Technology

Silica membrane

Main sample type

Human tissue

Sample amount

  • Up to 20 mg tissue fixed and (optionally) stabilized with PAXgene Tissue reagents
  • Up to 10 mg PF* tissue with high DNA content
  • 2–5 sections (5–10 µm thick) PFPE** tissue

Elution volume

14–40 µl

Time per run

30 min + 120 min incubation/8 samples

Processing

  • Manual: centrifugation
  • Automated: QIAcube

* PAXgene Tissue-fixed
** PAXgene Tissue-fixed, paraffin-embedded


Intended Use

The PAXgene Tissue DNA Kit is intended for the purification of DNA from PAXgene Tissue-fixed (PF) and PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissue samples. The kit is intended to be used as part of the PAXgene Tissue System, which additionally includes the PAXgene Tissue FIX Container and the PAXgene Tissue STABILIZER Concentrate for the collection, fixation, stabilization and storage of tissue samples.
 

For research use only. Not for use in diagnostic procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease. The performance characteristics of this product have not been fully established.

Order information

Product

Catalog Nr.

Price

PAXgene Tissue DNA Kit (50)

For 50 DNA preps: PAXgene DNA Mini Spin Columns, Processing Tubes, Microcentrifuge Tubes, Carrier RNA, and Buffers. To be used in conjunction with PAXgene Tissue FIX Containers + PAXgene Tissue STABILIZER reagent.

767134
289 EUR

Details

Performance

Total DNA isolated with the PAXgene Tissue DNA Kit is highly pure. DNA has A260/A280 ratios of 1.7–1.9, and absorbance scans show a symmetrical peak at 260 nm, confirming high purity of genomic DNA. Contamination is minimal and purified DNA is ready to be used in downstream applications with no detectable inhibition of PCR (Figure 1 and Figure 2)

Principle

The PAXgene Tissue DNA Kit enables purification of DNA from tissues fixed and stabilized using the PAXgene Fixation and Stabilization reagents, which preserve tissue morphology and biomolecule integrity by avoiding destructive crosslinking and degradation found in formalin-fixed tissues. The purified DNA has no inhibitory chemical modifications and thus, can be used in sensitive downstream applications.

Procedure

PAxgene Tissue-fixed (PF) or PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissue samples are lysed in Buffer TD1 with proteinase K. Conditions are adjusted for optimal binding of DNA to the silica membrane and the lysate is loaded on the PAXgene DNA spin column. DNA binds to the membrane while contaminants pass through. Enzyme inhibitors are effectively removed with subsequent washes and pure DNA is eluted in a low-salt elution buffer (Figure 3).

The PAXgene Tissue DNA Kit provides 2 protocols for purification of DNA from different starting materials:

  • Sections of PFPE tissue
  • PF tissue samples (without paraffin embedding)
Automation

DNA purification using the PAXgene Tissue DNA Kit can be automated on the QIAcube, enabling:

  • Elimination of manual processing steps
  • Elimination of up to 75% hands-on time compared to manual processing (Figure 4
  • Processing of up to 12 samples per run
  • Processing of normal, fibrous or fat-rich PAXgene Tissue-fixed samples (Figure 5)
  • Processing of 2–5 sections of PFPE tissue samples (Figure 8)

Two protocols are available for automated purification from different starting materials:

Applications

The purified DNA is ready to use in a wide range of downstream applications (see Resources), including:

  • PCR, multiplex, long-range, and quantitative, real-time PCR
  • Southern blotting
  • SNP genotyping
  • Next-generation sequencing (NGS)
(A) Hematoxylin and eosin (H&E) staining of PAXgene Tissue-fixed, paraffin-embedded (PFPE) human colorectal cancer tissue and (B) DNA on agarose gel electrophoresis using 0.8 % TBE buffered gels with 200 ng genomic DNA isolated in triplicate from five cases (#1–5) of human PFPE colorectal cancer. M: markers. Zoom image

Figure 1. High-quality DNA from PFPE tissue with preserved morphology.

(A) Hematoxylin and eosin (H&E) staining of PAXgene Tissue-fixed, ...
Multiplex and long-range PCR of DNA from PAXgene Tissue-fixed, paraffin-embedded (PFPE) human colorectal cancer tissue (modified according to Viertler et al. 2012). (A) Multiplex PCR of 8 different genomic DNA fragments ranging from 222 to 955 bp. (B) Long-range PCR of a 5 kb genomic DNA fragment. Zoom image

Figure 2. DNA without chemical modifications can be used for demanding downstream applications.

Multiplex and long-range PCR of DNA from PAXgene Tissue-fixed, paraffin-embedded ...
Tissue sample lysis is performed in lysis buffer, Buffer TD1, with proteinase K digestion. Binding conditions are adjusted with Buffer TD2 and ethanol to provide optimal DNA-binding conditions, and the lysate is loaded onto a PAXgene DNA spin column. During centrifugation, DNA is selectively bound to the silica membrane, and contaminants pass through. Remaining contaminants and enzyme inhibitors are removed in two efficient wash steps with wash buffers TD3 and TD4. DNA is then eluted in low-salt elution Buffer TD5 and is ready for use. Zoom image

Figure 3. The PAXgene Tissue DNA Procedure.

Tissue sample lysis is performed in lysis buffer, Buffer TD1, with proteinase K ...
Comparison of sample preparation times between manual and QIAcube procedures for 12 PAXgene Tissue-fixed, paraffin-embedded (PFPE) samples. Hands-on time without incubation times. Zoom image

Figure 4. Significant hands-on time savings compared to manual purification.

Comparison of sample preparation times between manual and QIAcube procedures for ...
PAXgene Tissue fixed (PF) tissue types, processed on the QIAcube. Twelve different PAXgene Tissue-fixed (PF) rat tissue types, including fibrous or fatty, DNA-rich and DNA-poor tissue, processed with the PAXgene Tissue DNA Kit on the QIAcube in one single run. DNA yield per 10 mg tissue. Zoom image

Figure 5. Yield of high-quality, high molecular weight DNA from 12 different PAXgene Tissue fixed (PF) tissue types, processed on the QIAcube.

PAXgene Tissue fixed (PF) tissue types, processed on the QIAcube. Twelve different ...
Twelve different PAXgene Tissue-fixed (PF) rat tissue types, including fibrous or fatty, DNA-rich and DNA-poor tissue, processed with the PAXgene Tissue DNA Kit on the QIAcube in one single run. Ratio of absorbance at 260 and 280 nm. Zoom image

Figure 6. Absorbance ratio of high-quality, high molecular weight DNA from 12 different PAXgene Tissue fixed (PF) tissue types, processed on the QIAcube.

Twelve different PAXgene Tissue-fixed (PF) rat tissue types, including fibrous or ...
Twelve different PAXgene Tissue-fixed (PF) rat tissue types, including fibrous or fatty, DNA-rich and DNA-poor tissue, processed with the PAXgene Tissue DNA Kit on the QIAcube in one single run. DNA from each tissue type (300 ng) on agarose gel electrophoresis using 1 % TBE buffered gels; M: markers. Zoom image

Figure 7. Agarose gel electrophoresis of high-quality, high molecular weight DNA from 12 different PAXgene Tissue fixed (PF) tissue types, processed on the QIAcube.

Twelve different PAXgene Tissue-fixed (PF) rat tissue types, including fibrous or ...
DNA yield from 3 sections (thickness 10 µm) each of 8 different PAXgene Tissue-fixed, paraffin-embedded (PFPE) rat tissue types processed in triplicate with the PAXgene Tissue DNA Kit on the QIAcube or manually. Zoom image

Figure 8. Comparison of manual and automated procedure: DNA yield from sections of PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissue.

DNA yield from 3 sections (thickness 10 µm) each of 8 different PAXgene ...
Ratio of absorbance at 260 and 280 nm from 3 sections (thickness 10 µm) each of 8 different PAXgene Tissue-fixed, paraffin-embedded (PFPE) rat tissue types processed in triplicate with the PAXgene Tissue DNA Kit on the QIAcube or manually. Zoom image

Figure 9. Comparison of manual and automated procedure: Absorbance ratio from sections of PAXgene Tissue fixed, paraffin-embedded (PFPE) tissue.

Ratio of absorbance at 260 and 280 nm from 3 sections (thickness 10 µm) each of 8 ...
Agarose gel electrophoresis from 3 sections (thickness 10 µm) each of 8 different PAXgene Tissue-fixed, paraffin-embedded (PFPE) rat tissue types processed in triplicate with the PAXgene Tissue DNA Kit on the QIAcube or manually. DNA from each replicate and tissue type (200 ng) on agarose gel electrophoresis using 1 % TBE buffered gels; M: markers. Zoom image

Figure 10. Comparison of manual and automated procedure: Agarose gel electrophoresis from sections of PAXgene Tissue fixed, paraffin-embedded (PFPE) tissue.

Agarose gel electrophoresis from 3 sections (thickness 10 µm) each of 8 different ...

Resources

PAXgene Tissue DNA Kit

Purification and quality of biomolecules from PAXgene Tissue-treated samples

1. Is it possible to use formalin-fixed, paraffin-embedded (FFPE) kits and protocols to isolate biomolecules from PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissues?
No. Procedures developed for the extraction of biomolecules from FFPE tissues include prolonged proteinase K digestion and heating steps to remove chemical modifications introduced by formalin. Since the PAXgene Tissue System does not chemically modify biomolecules, these steps are not necessary and, in fact, lead to degradation of biomolecules. Instead, use dedicated PAXgene Tissue Kits and supplementary protocols for extraction of biomolecules from PAXgene Tissue-treated samples.


2. What is the purity of nucleic acids extracted with the PAXgene Tissue Kits?
The PAXgene Tissue DNA and RNA, including miRNA, Kits are based on proven QIAGEN technologies. Nucleic acids isolated with these kits are generally of high purity.

On average, measurements of the A260/A280 ratio for DNA purified with the PAXgene Tissue DNA Kit are >1.7, and ratios for RNA, including miRNA, purified with the PAXgene Tissue RNA/miRNA Kit are >1.8.

3. How well is DNA integrity preserved in PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissues?  
In contrast to DNA isolated from formalin-fixed, paraffin-embedded (FFPE) tissue, DNA from PFPE tissue exhibits high molecular weight. In most cases, a distinct 10 kb band is observed in electrophoretically separated DNA eluates. For an example, see Figure 2 in the Technical Note "Quantitative analysis of KRAS and BRAF mutational status in DNA from PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissue using Pyrosequencing technology" under Resources.   

4. What are the yield and integrity of nucleic acids isolated from blocks of PAXgene Tissue-fixed, cryo-embedded (PFCE) tissues?    
DNA and RNA isolated from PFCE tissue specimens are of high quantity and quality, comparable to PFPE tissue.    


5. Are special kits and protocols required for the isolation of biomolecules from PAXgene Tissue fixed, cryo-embedded (PFCE) tissues?    
No. Regular PAXgene Tissue Kits can be used for the isolation of RNA, miRNA and DNA from PFCE tissue. Supplementary protocols developed specifically for the extraction of biomolecules from PFCE samples are available under Resources.


6. Which kits and protocols can be used for the isolation of nucleic acids from microdissected PAXgene-Tissue fixed, paraffin-embedded (PFPE) and PAXgene Tissue-fixed, cryo-embedded (PFCE) specimens?    
Regular PAXgene Tissue Kits can be used for the isolation of RNA, miRNA and DNA from microdissected PFPE and PFCE tissue. Supplementary protocols developed specifically for the extraction of biomolecules from microdissected PFPE and PFCE samples are available under Resources.    


Molecular analysis of biomolecules purified from PAXgene Tissue-treated samples    

1. What is the PCR performance of DNA purified from PAXgene Tissue-fixed, paraffin-embedded (PFPE) and PAXgene Tissue-fixed, cryo-embedded (PFCE) tissues compared to RNA from snap-frozen or formalin-fixed, paraffin-embedded (FFPE) tissues?    
In contrast to FFPE, DNA purified from PFPE and PFCE is of high molecular weight and free of chemical modifications. In demanding downstream applications, such as multiplex or long-range PCR, it performs similarly or identically to DNA isolated from frozen tissue. For examples, see, Figure 5 in Viertler et al. 2012  under Resources.

2. Is DNA purified from PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissues suitable for targeted NGS analysis?
Yes. DNA purified from PFPE is of high molecular weight and free of chemical modifications. Several independent researchers successfully used DNA from PFPE for NGS with different sample types, workflows and platforms (data in preparation). The data confirm recent suggestions that some DNA sequence artifacts associated with FFPE can be avoided with PFPE (see Högnäs et al. 2017 under Resources).

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