FFPE Tissue RNA Extraction Kit(SpinColumn)
Packing Specification：
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R2125-50
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FFPE Tissue RNA Extraction Kit(SpinColumn)
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50T
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CNY1800
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R2125-200
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FFPE Tissue RNA Extraction Kit(SpinColumn)
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200T
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CNY6480
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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 total RNA from formalin-fixed, paraffin-embedded (FFPE) tissue samples. The unique lysis buffer/Proteinase K rapidly lyses cells to release RNA. The lysis mixture is then passed through a genomic DNA removal column, where genomic DNA is retained while RNA passes through. After adjusting the binding conditions of the filtered RNA with ethanol, RNA is selectively adsorbed to the silica matrix membrane in the spin column under high chaotropic salt conditions. Through a series of rapid washing and centrifugation steps, deproteinization buffer and wash buffer remove impurities such as cellular metabolites and proteins. Finally, pure RNA is eluted from the silica matrix membrane with low-salt RNase-free H₂O. The obtained RNA can be used in reverse transcription PCR (RT-PCR), quantitative real-time PCR, and other experiments.
Product Features:
1.No toxic reagents such as phenol or chloroform are used, and no ethanol precipitation steps are required.
2.Fast and simple: RNA extraction for a single sample can generally be completed within 1 hour.
3.The kit's exclusive genomic DNA removal column and formula ensure effective elimination of genomic DNA residue. In most cases, the obtained RNA does not require DNase digestion and can be directly used in RT-PCR, quantitative real-time PCR, and other experiments.
4.Multiple column washing steps ensure high RNA purity, which is suitable for various downstream experiments directly.
Experimental Procedures:
Note: Before first use, add the indicated amount of absolute ethanol to the Wash Buffer RW bottle, mix thoroughly, and mark with a check in the box immediately after adding to avoid repeated addition!
1.Trim off excess paraffin outside the embedded tissue and cut into 5-20 μm thick sections (discard the first 2-3 sections).
2.Collect paraffin sections with a total thickness not exceeding ■40 μm into a 1.5-2 mL centrifuge tube (e.g., 2 sections of 20 μm, 4 sections of 10 μm, or 8 sections of 5 μm), or paraffin sections with a total thickness not exceeding ▲80 μm into a 2 mL centrifuge tube.
■ indicates the total thickness of processed sections ≤ 40 μm; ▲ indicates the total thickness of processed sections ≤ 80 μm.
3.Add 1 mL of 100% xylene, vortex for 10 seconds. Centrifuge briefly to immerse the tissue completely in xylene.
4.Incubate in a 50°C water bath for 3 minutes to melt the paraffin, then centrifuge at maximum speed at 20-25°C for 2 minutes to pellet the tissue at the bottom of the tube.
5.Carefully aspirate and discard the xylene supernatant with a pipette, being careful not to aspirate the pellet.
6.Add 1 mL of absolute ethanol, vortex, centrifuge at maximum speed for 2 minutes, and carefully aspirate and discard the ethanol supernatant.
7.Add 1 mL of absolute ethanol, repeat Step 6, and aspirate as much ethanol as possible.
8.Air-dry the ethanol at room temperature or 37°C for 10 minutes or until all ethanol has evaporated.
Complete ethanol evaporation is crucial; even trace ethanol residue can 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 pipette to mix well.
10.Incubate at 55°C for 15 minutes, then at 80°C for 15 minutes.
After incubation at 55°C, remove the centrifuge tube and place it at room temperature. Wait for the water bath to heat up to 80°C before placing the tube back for precise 15-minute incubation. Even a 2-minute extension may cause partial RNA degradation.
11.Add ■320 μL / ▲500 μL of Binding Buffer RBC, and pipette thoroughly to mix and adjust binding conditions.
12.Immediately add the mixture to a genomic DNA removal column (place the column into a collection tube), centrifuge at 14,000 rpm for 60 seconds, and retain the filtrate (RNA is in the filtrate).
Avoid aspirating large undigested flocculent substances onto the column to prevent clogging.
13.Add ■720 μL / ▲1200 μL of absolute ethanol to the filtrate, immediately pipette to mix well; do not centrifuge.
14.Immediately transfer the mixture (less than 700 μL each time, can be added in multiple portions) to an RNA Spin Column (SpinRA) (place the spin column into a collection tube), centrifuge at 13,000 rpm for 30 seconds, and discard the waste liquid.
15.Add 500 μL of Wash Buffer RW (please check if absolute ethanol has been added first!), centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid. Repeat this step with another 500 μL of Wash Buffer RW.
16.Place the SpinRA column back into the empty collection tube, centrifuge at 13,000 rpm for 2 minutes to remove as much wash buffer as possible, avoiding residual ethanol in the wash buffer that may inhibit downstream reactions.
17.Remove the SpinRA column, place it into a new RNase-free centrifuge tube. According to the expected RNA yield, add 30 μL of RNase-free water to the center of the adsorption membrane (heating the water in a 70-90°C water bath in advance can improve yield), incubate at room temperature for 1 minute, and centrifuge at 12,000 rpm for 1 minute.
Repeating the elution once results in a high-concentration RNA eluate. Combining two separate eluates increases the RNA yield by 15–30% compared to a single elution, but the concentration is lower. Users can choose according to their needs.
