FFPE RNA Purification

Main components

• Low RNA binding microcentrifuge tubes or 96 deep well plate
• Magnetic separation devices (for 15ml and 96 well microtiter plate)
• Absolute ethanol, 85% Ethanol and isopropanol
• Tube shaker / vortexer for 96 well plate and microcentrifuge tubes.

• Store all buffers at room temperature (15-30°C) and away from light.
• FFPE RNA Beads and FFPE RNA Beads can be stored at room temperature (15-30℃) when unopened, after opening, store in a refrigerator (2-8℃).
• DNse Ⅰ dry powder should be stored in a refrigerator (2-8°C) before adding DNase Dissolve Buffer; after adding DNase Dissolve Buffer, aliquot and store at -20°C, avoid repeated freezing and thawing.
• The kit content is stable for 12 months after the manufacture date.

Reagent Preparation

• Preparation of DNse Ⅰ solution: Dissolve DNase Ⅰ dry powder in the corresponding volume of DNase I Dissolve Buffer and mix gently. The dissolved DNase I solution should be aliquoted and stored at -20°C. The dissolved DNase I solution can be stored at 4°C for up to 6 weeks. Do not freeze and thaw.

• Prepare 85% ethanol solution.
• Before use, please check if there are any crystals precipitate in the RNA Binding Buffer (Buffer LWA). If there are crystals, redissolve the crystals in a 37°C water bath.
• Preparation of 1× RNA Washing Buffer (Buffer FWR1) according to table below (mark the box on the label after adding absolute ethanol).

• Preparation of 1× RNA washing buffer (Buffer SPW), add absolute ethanol as indicated in the table below and store at room temperature in the dark (mark the box on the label after adding absolute ethanol).

• Preparation of 1 × RNA Binding Buffer (Buffer FBR), mark the box on the label after adding absolute ethanol.

• FFPE sections: Take 5-8 FFPE tissue sections. The thickness of each section is between 5-10 μm and the surface area is 1×1 cm2.
• FFPE tissue block: Take about 20 mg of FFPE tissue and use a sterile scalpel to remove excess paraffin outside the selected sample area.
• Sample fixed in formaldehyde: Take about 20 mg of sample, cut it into several pieces with a scalpel, then put the sample into a 1.5-2ml centrifuge tube, add 400μl PBS (10 mM, pH7.4), centrifuge at 12000 rpm for 1 min, repeat 3 times.

RNA Isolation Procedures

1. Cut the FFPE sample into 5-10 μm thick sections, take 4-8 slices into a 1.5 mL or 2 mL microcentrifuge tube, add 300 μL Lysis Buffer (Buffe FLR) and 10 μL proteinase K (20mg/mL), vortex to mix, and incubate at 56°C for 15 minutes.
2. Incubate at 80°C for 15 minutes. then centrifuge briefly and transfer all the supernatant to a new 1.5 mL-2 mL centrifuge（≥12000 rpm）5 min.
3. Take 200 μL supernatant to a new microcentrifuge tube, add 400 μL of 1× Buffer FBR , 250 μL Absolute Alcohol and 40 μL of FFPE RNA beads, vortex to mix, and incubate at room temperature for 5 minutes, mix every 2-3 minutes to aid lysis and binding. Note: Magnetic beads tend to sink to the bottom, please resuspend the FFPE RNA beads by vortex for 1 minute, then add 30μl of FFPE RNA beads.
4. Place the microcentrifuge tube on a magnetic stand for about 30 seconds or until the solution clears, turn the tube over a few times while on the magnetic stand to remove the FFPE RNA beads trapped on the lid. With the sample tube on the magnetic stand, carefully aspirate the cleared supernatant without aspirating the FFPE RNA beads.
5. Add 700 μL of 1× Buffer FWR1, and vortex for 1-2 minutes to completely resuspend the magnetic beads.
6. Place the microcentrifuge tube on a magnetic stand for about 30 seconds or until the solution clears, turn the tube over a few times while on the magnetic stand to remove the FFPE RNA beads trapped on the lid. With the sample tube on the magnetic stand, carefully aspirate the cleared supernatant without aspirating the FFPE RNA beads.
7. Add 140 μL DNase Reaction Buffer (Buffer FWR2) and 6 μL DNase I (DNase I), mix gently with a pipette, centrifuge briefly to collect the solution to the bottom of the microcentrifuge tube, and incubate at room temperature for 20 minutes.
8. Place the microcentrifuge tube on a magnetic stand for about 30 seconds or until the solution clears, turn the tube over a few times while on the magnetic stand to remove the FFPE RNA beads trapped on the lid. With the sample tube on the magnetic stand, carefully aspirate the cleared supernatant without aspirating the FFPE RNA beads.
9. Remove the microcentrifuge tube from the magnetic separation rack, add 800 μL of 85% ethanol to wash, and vortex for 1-2 minutes to completely resuspend the magnetic beads.
10. Place the microcentrifuge tube on a magnetic stand for about 30 seconds or until the solution clears, turn the tube over a few times while on the magnetic stand to remove the FFPE RNA beads trapped on the lid. With the sample tube on the magnetic stand, carefully aspirate the cleared supernatant without aspirating the FFPE RNA beads.
11. Repeat steps 9 and 10.
12. 12. Pulse down and aspirate again to remove as much of the liquid as possible. Keeping the microcentrifuge tube on the magnet, air-dry the FFPE RNA beads at room temperature for 10 minutes. Note: Do not vacuum dry, excessive drying will reduce recovery rate.
13. Add at least 50 μL of RNA Eluent Buffer (RNase-Free H2O), vortex or pipet 10 times to fully resuspend the magnetic beads and incubate with constant shaking at 45°C for 5 minutes.
14. Place sample tubes on a magnetic stand, the solution will clear in about 1 minute. Transfer the cleared supernatant into a low RNA binding microcentrifuge tube. This is the purified RNA. Freeze the eluted RNA until you are ready for your downstream analysis