Products

FFPE Tissue microRNA Extraction Kit(SpinColumn)

Packing Specification：

For research use only. Not for use in medicine, clinical diagnosis, food, cosmetics or other applications.

Product Introduction:
This kit is designed for the rapid extraction of RNA (including microRNA) from formalin-fixed, paraffin-embedded (FFPE) tissue samples. The unique lysis buffer/Proteinase K rapidly lyses cells to release RNA. The lysate mixture is then passed through a Genomic DNA Removal Column, where genomic DNA is retained while RNA passes through as filtrate. After adjusting the binding conditions with ethanol, RNA selectively binds to the silica matrix membrane in the spin column under high chaotropic salt conditions. Through a series of rapid washing-centrifugation steps, impurities such as cellular metabolites and proteins are removed by the deproteinization solution and wash buffer. Finally, pure RNA is eluted from the silica matrix membrane using low-salt RNase-free H₂O. The obtained RNA can be used for reverse transcription PCR (RT-PCR), quantitative real-time PCR (qPCR), and other experiments.

Product Features:
1.The silica matrix membranes in the spin columns are all high-quality specially manufactured adsorption membranes, ensuring minimal variation in adsorption capacity between columns.
2.Eliminates the need for toxic reagents such as phenol and chloroform, as well as ethanol precipitation steps.
3.Rapid and simple operation: RNA extraction from a single sample can generally be completed within 1 hour.
4.The kit’s exclusive genomic DNA removal column and formulation ensure effective elimination of residual genomic DNA. In most cases, the extracted RNA does not require DNase digestion and can be directly used for RT-PCR, qPCR, and other experiments.
5.Multiple column washing steps ensure high RNA purity, with a typical OD260/OD280 ratio of 2.1-2.2 (the ratio of 100% pure RNA is generally around 2.2). The RNA can be directly used for various downstream experiments.

Application Scope:
Suitable for the rapid extraction of RNA (including microRNA) from formalin-fixed, paraffin-embedded tissue samples. The extracted RNA can be directly used for RT-PCR and qPCR.

Operation Steps:
Note: Before first use, add the specified volume of absolute ethanol to Wash Buffer RW, mix thoroughly. Mark the corresponding box immediately after adding ethanol to avoid repeated additions!

1.Trim excess paraffin surrounding the embedded tissue and cut into 10-20 μm thick sections.
Note: Sections must be ≥10 μm thick; excessively thin sections may disrupt cells, leading to microRNA loss during dewaxing.

2.Collect paraffin sections with a total thickness of ≤■40 μm into a 1.5-2 mL centrifuge tube (e.g., 2 sections of 20 μm or 4 sections of 10 μm) or ≤▲80 μm into a 2 mL centrifuge tube.
■ Indicates total section thickness ≤40 μm; ▲ Indicates total section thickness ≤80 μm.

3.Add 1 mL of 100% xylene, vortex for 10 seconds. Centrifuge briefly to submerge the tissue completely in xylene.

4.Incubate in a 50℃ water bath for 3 minutes to melt the paraffin. Centrifuge at maximum speed at 20-25℃ for 2 minutes to pellet the tissue.

5.Carefully aspirate and discard the xylene supernatant, taking care not to disturb the pellet.

6.Add 1 mL of absolute ethanol, vortex, centrifuge at maximum speed for 2 minutes. Carefully aspirate and discard the ethanol supernatant.

7.Repeat Step 6 with another 1 mL of absolute ethanol to remove residual xylene. Aspirate as much ethanol as possible.

8.Air-dry the ethanol at room temperature or 37℃ for 10 minutes or until all ethanol is completely volatilized.
Note: Complete ethanol evaporation is critical; trace ethanol residue will reduce RNA yield.

9.Resuspend the tissue pellet thoroughly in ■150 μL / ▲240 μL of Lysis Buffer PKD by pipetting or vortexing. Centrifuge briefly to collect the liquid at the bottom of the tube. Add 10 μL of Proteinase K and mix by pipetting.

10.Incubate at 55℃ for 15 minutes, then at 80℃ for 15 minutes.
Note: After incubation at 55℃, remove the centrifuge tube and allow it to cool to room temperature. Place it back in the water bath once the temperature reaches 80℃, and incubate for exactly 15 minutes. Even a 2-minute extension may cause partial RNA degradation.

11.Add ■320 μL / ▲500 μL of Binding Buffer RBC, mix thoroughly by pipetting to adjust binding conditions.

12.Immediately load the mixture into a Genomic DNA Removal Column (placed in a collection tube). Centrifuge at 14,000 rpm for 30 seconds and retain the filtrate (RNA is in the filtrate).
Note: Avoid loading large undigested clumps onto the column to prevent clogging.

13.Add ■1120 μL / ▲1750 μL of absolute ethanol to the filtrate, mix immediately by pipetting. Do not centrifuge.
Note: If adding 1750 μL of absolute ethanol, first transfer the filtrate to a centrifuge tube with a volume exceeding 3 mL before adding ethanol.

14.Load the mixture into a SpinRA RNA Adsorption Column (placed in a collection tube) in batches (≤700 μL per aliquot). Centrifuge at 13,000 rpm for 30 seconds and discard the filtrate.

15.Add 500 μL of Wash Buffer RW (check if absolute ethanol has been added!). Centrifuge at 12,000 rpm for 30 seconds and discard the filtrate. Repeat this step with another 500 μL of Wash Buffer RW.

16.Place the SpinRA Adsorption Column back into the empty collection tube. Centrifuge at 13,000 rpm for 2 minutes to completely remove residual wash buffer (residual ethanol may inhibit downstream reactions).

17.Transfer the SpinRA Adsorption Column to a new RNase-free centrifuge tube. Add 30 μL of RNase-free water (heating in a 70-90℃ water bath can improve yield) to the center of the adsorption membrane according to the expected RNA yield. Incubate at room temperature for 1 minute, then centrifuge at 12,000 rpm for 1 minute.
Higher elution volume leads to higher elution efficiency. To obtain higher RNA concentration, the elution volume can be appropriately reduced. Alternatively, reapply the eluate to the SpinRA Adsorption Column for a second elution.