About Pinpoint™ Slide RNA Isolation System II
The Pinpoint™ Slide RNA Isolation System II is an innovative product for the isolation of RNA from any targeted area of fresh or paraffin-embedded tissue sectioned onto a glass slide. The system combines powerful Pinpoint™ tissue sampling methodology, a unique single-step RNA extraction/binding buffer, and Fast-Spin column purification technology to yield high quality RNA. Unlike current UV-based methods, this product makes isolation of tissue RNA simple and quick. No expensive specialized equipment is needed. Eluted RNA is well suited for subsequent RNA analyses including RT-PCR.
| Format | Spin Column. |
|---|---|
| Processing Time | 5 hr |
| Equipment | Microcentrifuge |
| Sample Sources | Cells from tissue sections on glass slides. |
| RNA Recovery | Typically, up to 5 µg RNA is eluted into ≥ 8 µl RNase/DNase-free water allowing for a highly concentrated sample. |
| RNA Storage | Recommended that 1 U/10 µl RNase inhibitor be added to the RNA prior to storage at -70°C. |
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RT-PCR of RNA recovered from human tissue using the Pinpoint™ RNA Isolation System. Amplicons (in duplicate) are from A) a human ß-actin transcript; B) an arbitrary human transcript from Chromosome 3. M is 100 bp DNA Marker (Zymo Research).
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I. Paraffin Removal from the Tissue Sample 1. Mount the paraffin-embedded tissue section (≥10 µm thick) onto a glass slide and dry it at 60°C for 30 minutes. 2. Submerge the slide in xylene at room temperature for 1 hour changing the xylene once after 30 minutes. 3. Hydrate the sample by washing progressively for 2 minutes in 100%, 70%, 50% ethanol, and then pure water. 4. Air-dry the sample on the slide. RNA isolation using the Pinpoint™ Slide RNA Isolation System II can now be performed.
II. Pinpoint Fractionation (Procedure for the removal of a selected area of tissue from a glass slide.) 1. Apply the Pinpoint™ Solution1 to the area of tissue on the slide where the RNA is to be extracted2. Use a sterile pipette tip or a syringe to gently spread a small amount of Pinpoint™ Solution over the selected tissue region. Generally, use about 0.5 µl of Pinpoint™ Solution per mm2 of tissue area 2. Allow the Pinpoint™ Solution to dry completely at room temperature. (Usually about 30 to 45 minutes). The PinpointTM Solution should dry as a blue film embedding the tissue and cells underneath. 3. Remove the embedded tissue from the slide. Use a sterile blade or scalpel to cut, and then remove the embedded section from the slide. Transfer the sample to a 1.5 ml tube. 4. Centrifuge briefly to locate the tissue sample at the bottom of the tube. III. RNA Extraction (Procedure for the extraction and purification of total RNA from a deparaffinized tissue sample.) 1. Add 20 µl of RNA Digestion Buffer and 5 µl Proteinase K to the tube containing the recovered tissue. Mix gently. For multiple samples, the RNA Digestion Buffer and Proteinase K may be premixed. Add 25 µl of this mixture to each sample. 2. Incubate the tubes at 55°C for 4 hours. 3. Centrifuge the tubes briefly when the incubation is finished. 4. Add 50 µl (2 volumes) of RNA Extraction Buffer and mix. 5. Add 75 µl (1 volume) of 95-100% ethanol to the tube. Lightly vortex. 6. Transfer the mixture to the Zymo-Spin™ IC Column in a Collection Tube. 7. Spin the column at ≥10,000 x g for 1 minute. 8. Add 200 µl RNA Wash Buffer to the Zymo-Spin™ IC Column and centrifuge at ≥10,000 x g for 1minute. Discard flow-through. Repeat this step. 9. Transfer the column into a new RNase-Free Tube. 10. Add 10 µl of prewarmed DNase/RNase-Free Water (60°C) directly to the column matrix. Wait for 2 minutes then centrifuge at ≥10,000 x g for 1 minute and collect the eluted RNA. The RNA can be used immediately or stored at -70 oC. |
