The FFPE microRNA Isolation Kit is constructed to isolate total RNA and microRNA (20-200 nt, such as miRNA, siRNA, and snRNA) from formalin-fixed, paraffin-embedded tissues in a quick, convenient, and cost-effective manner. Following the removal of gDNA, the flow-through is infused with a higher concentration of ethanol to enhance RNA (including microRNA) binding affinity to the RNA Column membrane. The bound RNA molecules are then eluted, making them ready for molecular biological experiments like RT-PCR, Real-time RT-PCR, Northern Blot, total RNA sequencing, and microRNA analysis, among others. Proteinase K should be kept at a temperature of -20°C. The other ingredients can be kept for up to 9 months at room temperature (15-25°C).
Storage:
Proteinase K should be stored at -20°C. The other components can be stored at room temperature (15-25°C) for up to 9 months.
Components:
Specifications:
| Features | Efficiency: efficient enrichment of total RNA and microRNA (< 200 nucleotides). Simplity and convenience: the procedure is quick and convenient. High Performance: high-purity RNA suitable for molecular biological experiments. |
| Application | The purified RNA can be used directly for molecular biological experiments such as RT-PCR, Real-time RT-PCR, Northern Blot, total RNA sequencing, and microRNA analysis, etc. |
| Sample type | Formalin-fixed, paraffin-embedded tissues |
- Using a scalpel, trim excess paraffin off the sample block. Cut sections 10-20 µm thick. If the sample surface has been exposed to air, discard the first 2-3 sections.
- Immediately place the sections in a ■ 1.5-2 ml or ◆ 2 ml microcentrifuge tube (not supplied), and close the lid.
- Add 1ml 100% xylene, vortex vigorously 10 s, and centrifuge briefly to immerge the entire tissue in xylene.
- Incubate at 50°C for 3 min, centrifuge for 2 min at full speed at 20-25°C to bring the sample to the bottom of the tube.
- Remove the supernatant by pipetting.
- Add 1 ml ethanol (96-100%) to the pellet, and vortex for 10 s. Centrifuge for 2 min at full speed at room temperature. Remove the supernatant by pipetting.
- Repeat step 6.
- Open the tube and incubate at room temperature for 5-10 min or until all the residual ethanol has evaporated.
- Re-suspend the pellet in ■ 150 µL ◆ 240 µL Buffer PKD, centrifuge briefly to bring the liquid to the bottom of the tube. Add 10 µL Proteinase K, mix thoroughly.
- Incubate at 55 °C for 15 min, then 80 °C for 15 min .
- Add ■ 320 µL ◆ 500 µL Buffer RBC, and mix thoroughly by pipetting up and down several times.
- Transfer the mixture to gDNA Filter Column Set in a collection tube (supplied), centrifuge for 30 s at 14,000 rpm. Discard the gDNA Filter Column Set and keep the flow-through.
- Add ■ 1120 µL ◆ 1750 µL ethanol (96%-100%) to the flow-through and mix thoroughly by pipetting up and down several times. Do not centrifuge. Continue the next step without delay.
(2) A precipitate may form after addition of ethanol, but this will not affect the procedure. - Pipet up to 700 µl of the sample every time, including any precipitate that may have formed, into an RNA Column Set in a collection tube (supplied). Close the lid gently and centrifuge at 13,000 rpm for 30 s at room temperature. Discard the flow-through.
- Add 500 µl Buffer RW (Ensure that ethanol (96% -100%) has been added ) to the RNA Column Set. Close the lid gently and centrifuge for 30 s at 12,000 rpm to wash the column. Discard the flow-through.
- Repeat step 15.
- Centrifuge for 2 min at 13,000 rpm to dry the spin column membrane.
- Transfer the RNA Column Set to a new 1.5 ml collection tube (not supplied), and discard the old collection tube with the flow-through. Pipet 30 µl RNase-free H2O directly onto the RNA Column membrane. Close the lid gently. Place the tube on the benchtop at room temperature for 1 min, then centrifuge for 1 min at 12,000 rpm to elute the RNA.
Note: In the procedure, ■ indicates the total thickness of sections ≤ 40 µm, while ◆ indicates the the t otal thickness of sections > 40 µm.
