Cat. No. / ID: 767134
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.
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").
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.
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").
|Applications||PCR and quantitative, real-time PCR, Southern blotting, pharmacogenomic studies, SNP discovery and SNP genotyping|
|Main sample type||Human tissue|
|Time per run||30 min + 120 min incubation/8 samples|
|Elution volume||14–40 µl|
|Sample amount||4 x 15 x 15 mm|
|Yield||Depends on tissue type and starting material (fixed or PFPE*) * PAXgene Fixed Paraffin Embedded.|
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.
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.