Kit
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Kit Content
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Catalog No.
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PAXgene Tissue DNA Kit (50)
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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
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767134
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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.
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. 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 “Intact Genomic DNA and Improved PCR with PAXgene Tissue DNA”).
Sample collection and stabilization
PAXgene Tissue Containers are dual-chamber containers prefilled with 2 reagents (see figure “PAXgene Tissue Container 01, -02, -03, -04”). 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 (see figure “PAXgene Tissue Container Workflow”). 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 a minimum of 2 and a maximum of 4 weeks at 2–8°C, depending on the type of tissue.** Stabilized samples can be embedded in paraffin for histological studies (see the PAXgene Tissue Container Product Circular for information about tissue fixation, stabilization, processing, and paraffin embedding). With the PAXgene Tissue DNA kit, high-quality DNA can be isolated from sections of PAXgene Tissue fixed, paraffin-embedded (PFPE) tissue.
DNA purification
After deparaffination, 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”).
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.
Applications
The purified DNA is ready to use in a wide range of downstream research applications, including:
• PCR and quantitative, real-time PCR
• Southern blotting
• Pharmacogenomic studies
• SNP discovery and SNP genotyping
*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.
**Specifications for fixation and storage conditions in PAXgene Tissue Fix and PAXgene Tissue Stabilizer were determined using animal tissues.
