FFPE DNA/RNA Purification Kit

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 DNase 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. Note: Please follow the label instructions to add the corresponding volume of DNase Dissolve Buffer for 50 / kit and 100 / kit.
• Prepare 85% ethanol solution.
• Before use, please check if there are any crystals precipitate in the DNA 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× DNA 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.

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

1. Prepare the FFPE samples for RNA and DNA purification process:
2. 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.
3. FFPE tissue block: Take about 30 mg of FFPE tissue and use a sterile scalpel to remove excess paraffin outside the selected sample area.
4. Sample fixed in formaldehyde: Take about 30 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.
5. Cut the FFPE sample into 5-10 μm thick sections, take 4-8 slices into a 1.5 mL or 2 mL centrifuge tube, add 600 μL Lysis Buffer (Buffe FLR) and 30 μL proteinase K (Proteinase K), vortex to mix, and incubate at 56°C for 15 minutes.
6. Centrifuge at room temperature (≥12000 rpm) for 5 minutes. Take 300 uL of the supernatant for RNA purification process, and the remaining 300 uL of precipitation and supernatant for the DNA purification process.

DNA Extraction process

1. Aspirate the precipitate and remaining supernatant obtained in step 3 above, treat at 56°C for 2 hours, then at 90°C for 1 hour, and centrifuge at room temperature (≥12000 rpm) for 5 minutes.
2. Transfer all supernatant ≤300 μL to a new 1.5 mL-2 mL microcentrifuge tube.
3. Add 400 μL of DNA Binding Buffer (Buffer LWA) and 200 μL of isopropanol.
4. Vortex the FFPE DNA Beads for 1 minute to fully resuspend and add 20 μL FFPE DNA Beads to the microcentrifuge tube, vortex, or pipet 10 times to mix the FFPE DNA Beads thoroughly, incubate at room temperature for 10 minutes, shake for 1 minute every 3 minutes to help lysis and binding. Note: Magnetic beads tend to sink to the bottom, resuspend the FFPE DNA beads by vortexing for 1 minute, then add 20μl of FFPE DNA beads.
5. 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 DNA beads trapped on the lid. With the sample tube on the magnetic stand, carefully aspirate the cleared supernatant without aspirating the FFPE DNA beads
6. Remove the microcentrifuge tube from the magnetic separation stand, add 600 μL Washing Buffer (Buffer WB), and vortex for 1 min to completely resuspend the magnetic beads.
7. 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 DNA beads trapped on the lid. With the sample tube on the magnetic stand, carefully aspirate the cleared supernatant without aspirating the FFPE DNA beads.
8. Remove the microcentrifuge tube from the magnetic separation rack, add 600 μL of 1× Buffer SPW, vortex for 1 minute to completely resuspend the magnetic beads.
9. 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 DNA beads trapped on the lid. With the sample tube on the magnetic stand, carefully aspirate the cleared supernatant without aspirating the FFPE DNA beads.
10. Repeat steps 8 and 9.
11. 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 DNA beads at room temperature for 10 minutes.
    Note: Do not vacuum dry, excessive drying will reduce recovery rate.
12. Add at least 80 μL of DNA Elution Buffer (Buffer TE), vortex or pipet 10 times to fully resuspend the magnetic beads and incubate with shaking at 65°C for 10 minutes. Note: The elution effect depends on the pH of the eluent. Maximum elution efficiency is obtained at a pH between 7.0-8.5. Make sure the pH is within this range when eluting with water, and store the eluted DNA at -20°C.
13. Place the microcentrifuge tube on the magnetic separation rack. After the magnetic beads are completely adsorbed, transfer all the supernatant to a clean microcentrifuge tube and store eluted DNA under appropriate conditions.