PAXgene Tissue DNA Kit

For purification of DNA from tissues fixed and stabilized in PAXgene Tissue Containers

Features

  • Integrated fixation, stabilization, and purification
  • High-quality DNA from tissues
  • Preserved tissue morphology
PAXgene Tissue DNA Kit (50)

Cat. No. / ID: 767134

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 Containers
Specifications for fixation and storage conditions in PAXgene Tissue Fix and PAXgene Tissue Stabilizer were determined using animal tissues.
For Research Use Only. Not for use in diagnostics procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease.

Product Details

Tissue samples fixed and stored in PAXgene Tissue Containers can be paraffin-embedded and used for pathological studies as well as for subsequent purification of DNA, RNA, and/or miRNA. The PAXgene Tissue DNA Kit provides purification of DNA from tissues fixed and stabilized in PAXgene Tissue Containers. Purification is carried out using silica-based DNA purification technology in a spin-column format. Used with the containers, the kit provides a complete preanalytical solution for collection, fixation, and stabilization through to purification of high-quality DNA for molecular analysis. The procedures can be fully automated on the QIAcube Connect.

Performance

Total DNA purified using 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 the high purity of genomic DNA. Contamination is minimized, and purified DNA is ready to use in downstream applications with no detectable PCR inhibition. Together the container and kit provides a complete preanalytical solution for collection, fixation, and stabilization of tissue, and purification of high-quality DNA for molecular analysis (see figure "High-quality DNA from different tissue types fixed and stabilized in PAXgene Tissue Containers").

Principle

Current tissue fixation methods used in traditional histology are of limited use for molecular analysis. Fixatives that contain formaldehyde cross-link biomolecules and modify nucleic acids and proteins. During tissue fixation, storage, and processing, cross-links lead to degradation of nucleic acids. Since cross-links cannot be removed completely, the resulting chemical modifications can cause inhibition in sensitive downstream applications such as quantitative PCR or RT-PCR. In order to enable both molecular and traditional pathology testing from the same specimen, a method is needed for stabilization of molecular content and preservation of morphology.

PreAnalytiX has developed the PAXgene Tissue System to meet such needs. The system consists of a tissue collection device (the PAXgene Tissue Container for collection, stabilization, storage, and transportation of human tissue specimens) and kits for purification of DNA, total RNA, or miRNA. PAXgene Tissue Containers provide tissue fixation for histopathology studies and enable purification of high-quality nucleic acids from the same sample for molecular analysis. The fixation and stabilization method preserves tissue morphology and the integrity of nucleic acids without destructive cross-linking and degradation found in formalin-fixed tissues.

For isolation of genomic DNA, the system requires the use of PAXgene Tissue Containers for tissue collection and stabilization, followed by DNA isolation and purification using the PAXgene Tissue DNA Kit.

Procedure

PAXgene Tissue Containers are dual-chamber containers prefilled with 2 reagents. PAXgene Tissue Fix rapidly penetrates and fixes the tissue. After fixation, the tissue is removed from the PAXgene Tissue Fix and transferred to PAXgene Tissue Stabilizer in the second chamber of the same container. When the tissue is stored in PAXgene Tissue Stabilizer, nucleic acids and morphology of the tissue sample are stable for a minimum of 3 and a maximum of 7 days at room temperature or for up to 8 weeks at 2–8°C, depending on the type of tissue. Storage at –15 to –30°C is also possible for at least 26 months without any negative effects on the morphology of the tissue or the integrity of the nucleic acids.

Stabilized samples can be embedded in paraffin for histological studies. Nucleic acids can be isolated from the PAXgene Tissue fixed, paraffin-embedded (PFPE) samples either before or after embedding in paraffin. 

The PAXgene Tissue DNA Kit provides 3 protocols for purification of genomic DNA from tissues fixed and stabilized in PAXgene Tissue Containers.

Lysis of the tissue sample is performed in the lysis buffer, Buffer TD1, with digestion using proteinase K. Buffering conditions are adjusted with binding Buffer TD2 and ethanol to provide optimal DNA-binding conditions, and the lysate is loaded onto the 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 2 efficient wash steps with wash buffers TD3 and TD4, and DNA is then eluted in low-salt elution Buffer TD5, ready for use (see figure " The PAXgene Tissue DNA procedure").

See figures

Applications

The purified DNA is ready to use in a wide range of downstream applications, including:
  • PCR and quantitative, real-time PCR
  • Southern blotting
  • Pharmacogenomic studies
  • SNP discovery and SNP genotyping

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsPCR and quantitative, real-time PCR, Southern blotting, pharmacogenomic studies, SNP discovery and SNP genotyping
FormatSpin column
Main sample typeHuman tissue
ProcessingManual (centrifugation)
Time per run30 min + 120 min incubation/8 samples
Elution volume14–40 µl
Sample amount4 x 15 x 15 mm
YieldDepends on tissue type and starting material (fixed or PFPE*) * PAXgene Fixed Paraffin Embedded.
TechnologySilica technology

Resources

Brochures & Guides (3)
Two worlds in one sample
For isolation and purification of genomic DNA from tissue samples stabilized using the PAXgene Tissue System 

 

Moving toward excellence and standardization in tissue collection and fixation

 

Kit Handbooks (1)
For isolation and purification of genomic DNA from tissue samples stabilized in PAXgene Tissue Containers

Publications

Comprehensive DNA Methylation and Extensive Mutation Analyses of HER2-Positive Breast Cancer.
Yamaguchi T; Mukai H; Yamashita S; Fujii S; Ushijima T;
Oncology; 2015; 88 (6):377-84 2015 Jan 14 PMID:25591616
Next-gen tissue: preservation of molecular and morphological fidelity in prostate tissue.
Gillard M; Tom WR; Antic T; Paner GP; Lingen MW; VanderWeele DJ;
Am J Transl Res; 2015; 7 (7):1227-35 2015 Jul 15 PMID:26328007
Assessment of PAXgene Fixation on Preservation of Morphology and Nucleic Acids in Microdissected Retina Tissue.
Liu Y; Edward DP;
Curr Eye Res; 2016; 42 (1):104-110 2016 Jul 13 PMID:27409982
A Critical Evaluation of the PAXgene Tissue Fixation System:  Morphology, Immunohistochemistry, Molecular Biology, and Proteomics.
Mathieson W; Marcon N; Antunes L; Ashford DA; Betsou F; Frasquilho SG; Kofanova OA; McKay SC; Pericleous S; Smith C; Unger KM; Zeller C; Thomas GA;
Am J Clin Pathol; 2016; 146 (1):25-40 2016 Jul PMID:27402607
The focus on sample quality: Influence of colon tissue collection on reliability of qPCR data.
Korenkova V; Slyskova J; Novosadova V; Pizzamiglio S; Langerova L; Bjorkman J; Vycital O; Liska V; Levy M; Veskrna K; Vodicka P; Vodickova L; Kubista M; Verderio P;
Sci Rep; 2016; 6 :29023 2016 Jul 7 PMID:27383461

FAQ

Where can I find additional information for PreAnalytiX PAXgene products?
You can find additional information relating to the PreAnalytiX PAXgene products on the PreAnalytiX website .
FAQ ID - 3515
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 a special processing protocol needed?

To prevent biomolecule degradation during processing, dehydration must begin with at least 70–100% ethanol. We recommend using low-melting paraffin (melting point ≤56°C) and do not incubate samples in liquid paraffin for more than 3 hours.

Processing protocols for the PAXgene Tissue System are listed in the appendices of the PAXgene Tissue Container Product Circular and PAXgene Tissue FIX Container (50 ml) Product Circular.
FAQ ID -2523
What is the RT-PCR performance of RNA purified from PAXgene Tissue fixed, paraffin-embedded (PFPE) and PAXgene Tissue fixed, cryo-embedded (PFCE) tissue compared to RNA from snap frozen or formalin-fixed, paraffin-embedded (FFPE) tissue?
The PAXgene Tissue fixation and stabilization chemistry is free of cross-linking reagents. RNA purified from PFPE and PFCE is free of chemical modifications and performs similarly or identically to RNA isolated from frozen tissue. For examples of the correlation of gene expression levels in snap frozen tissue, FFPE, and PFPE, see Figure 4 in Groelz et al., Exp Mol Pathol. 2013 Feb; 94(1) and Figure 3 in Viertler et al., J Mol Diagn. 2012 Sep; 14(5).
FAQ ID -2538
What is the purity of nucleic acids extracted with the PAXgene Tissue kits?

The PAXgene Tissue DNA, RNA, and 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 and miRNA purified with the PAXgene Tissue RNA or miRNA Kits are both >1.8.

For examples of RNA purity see Technical Note "Yield, purity, and integrity of RNA purified from PAXgene Tissue fixed, paraffin-embedded (PFPE) rat tissue" under the Product Resources tab.

FAQ ID -2533
Is it possible to microdissect PAXgene Tissue fixed, paraffin-embedded (PFPE) and PAXgene Tissue fixed, cryo-embedded (PFCE) tissues?
Yes. Supplementary protocols for generating sections from PAXgene Tissue fixed, Paraffin-embedded (PFPE) and PAXgene Tissue fixed, cryo-embedded (PFCE) tissue blocks for manual and laser microdissection are available under the Product Resources tab.
FAQ ID -2531