Description
VitroSure™ DNA/RNA FFPE Tissue Isolation Kit is specially designed to optimize for the simultaneous isolation genomic DNA and total RNA (including small RNAs) from FFPE tissue sections. Pure DNA and RNA are released sequentially by differential solubilization of the same FFPE entire sample.
Kit Contents
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Technical Specifications
| Equipment needed | Microcentrifuge, heat block/bath (37°C, 56°C, 80°C and 90°C) |
| Type Isolated | Total DNA/RNA |
| Size Range | DNA> 50 bp/RNA> 70 bp |
| Yield | Up to 25 µg total DNA/RNA can be eluted into ≥ 50 µl/≥ 20 µl |
| Purity | Typical DNA: A260/A280 ≥ 1.8 /RNA: A260/A280 ≥ 2.0 |
| Eluted RNA Storage | at ≤ -20°C |
| Sample Source | Tissue from paraffin block or tissue sections |
| Processing Capacity | FFPE Tissue : ≤25 mg or 2-8 sections at thickness of 7-10 µm with a surface area of 15-20 mm2 |
| Applicable For | PCR and Next generation sequencing (NGS), genotyping,
Restriction enzyme digestion, SNP, etc. |
Storage
Store the Proteinase K Powder and DNase I powder at 2-8°C. After reconstitution of Proteinase K and DNase I store the solution at -20°C. The rest can be stored at room temperature.
Procedures
1. Sample preparation
1) Sample preparation from FFPE block:
- Use a scalpel to trim excess paraffin off the sample block.
- Cut up to 8 sections, each 5–10 µm thick. Discard the first 2–3 sections if the sample surface has been exposed to air.
- Place the sections immediately in a 1.5 or 2 ml microcentrifuge tube and add 1 ml of xylene to the sample. Close the lid and vortex vigorously for 10 seconds.
- Centrifuge at maximum speed for 2 minutes at room temperature.
- Carefully remove the supernatant without disturbing the pellets.
- Add 1 ml of ethanol (96–100%) to the pellet and mix by vortexing to extract residual xylene from the sample.
- Centrifuge at maximum speed for 2 minutes at room temperature.
- Carefully remove the supernatant without disturbing the pellet. Remove any remaining ethanol with a fine pipette tip.
- Open the tube and incubate at room temperature or up to 37°C for 10 minutes or until all residual ethanol has evaporated.
2) Sample preparation from FFPE sections on slides:
- Submerge the slides in xylene I for 3 minutes, followed by xylene II for an additional 3 minutes.
- Remove xylene by rinsing with 100% ethanol (1 minutes each, repeated twice).
- Air dry the slides for 3-5 minutes.
- Gently detach the tissue sections from the slides using a small blade, then transfer the tissue pellets into a 1.5 ml microcentrifuge tube.
2. Reconstitute the Proteinase K solution by combining 260 µl of Proteinase K buffer with 5 mg of Proteinase K powder. For the DNase I solution, add 550 µl of RNase-Free Water to 1 mg of DNase I powder, and gently mix by inverting the vial. After reconstitution, aliquot the Proteinase K and DNase I solutions, then store them at -20°C. Please refrain from subjecting the solutions to repeated thaw and melt cycles to maintain their stability.
3. Resuspend the pellets in 150 µl Buffer VKD. Add 10 µl of proteinase K solution and mix by vortexing.
4. Incubate at 56°C for 15 min and then incubate on ice for 3 min.
5. Centrifuge for 15 min at 20,000×g and carefully transfer the supernatant to a new microcentrifuge tube without disturbing the pellet for RNA purification.
6. Keep the pellet for DNA purification.
User Manual and Material Safety Data Sheet (MSDS) (PDF )
Note
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
Precautions
Handle with care. Avoid contact with eyes, skin and clothing. Do not ingest. Wear gloves.
