Description
VitroSure™ RNA FFPE Tissue Isolation Kit is optimized for isolation RNA from FFPE tissue samples and uses well-established VitroSure RNA Elute Columns for purification of total RNA from small sample volume size. This kit combines the selective binding properties of a silica-based membrane with flexible elution volumes of 15-30 µl.
Kit Contents
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Technical Specifications
| Equipment needed | Microcentrifuge, heat block/bath (37°C, 56°C and 80°C) |
| RNA Type Isolated | Total RNA |
| Size Range | > 70 bp |
| Yield | Up to 25 µg total RNA can be eluted into ≥ 20 µl |
| Purity | Typical A260/A280 ≥ 1.8 |
| Eluted RNA Storage | at ≤ -20°C |
| Sample Source | Tissue from paraffin block or FFPE 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 | RT-PCR, hybridization and Next generation sequencing (NGS), 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 270 µl of RNase-Free Water to 2 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.
6. Incubate at 80°C for 15 min.
7. Add 320 µl of Buffer VLT to the sample and mix thoroughly by vortexing.
8. Add 720 µl ethanol (96–100%) and mix well by vortexing or pipetting.
9. Carefully transfer the entire lysate to an RNA Elute column and centrifuge at 8000×g (or 10000 rpm) for 1 minute. Discard the flow-through. Repeat this step until complete sample is used.
10. Add 350 µl of Buffer VRN and centrifuge at 8000×g (or 10000 rpm) for 1 minute. Discard the flow-through.
11. Mix 10 µl DNase I solution with 70 µl Buffer VDD gently and add directly to the column membrane. Incubate at 20–30°C for 15 min.
12 Add 500 µl of Buffer VRN and centrifuge at 8000×g (or 10000 rpm) for 1 minute. Save the flow-through.
13. Place the column in a clean 2 ml collection tube. Apply the flow-through to the column and centrifuge at 8000×g (or 10000 rpm) for 1 minute. Discard the flow-through.
14. Add 500 µl Buffer VPE to the column. Centrifuge at 8000×g (or 10000 rpm) for 1 minute and discard the flow-through. Repeat this step once more.
15. Centrifuge at maximum speed for 3 minutes with the lid open to completely dry the memebrane.
16. Place the column in a clean 5 ml microcentrifuge tube and apply 10–30 µl RNase free water to the center of the membrane. Ensure that RNase free water is at room temperature.
17. Incubate for 1 min at room temperature (15–25°C) and centrifuge at full speed (20,000×g or 14,000 rpm) for 1 min to elute the RNA.
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.
