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.
|Processing Time||5 hr|
|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.|
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).
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.