Fibrous tissue RNA Extraction Kit(SpinColumn)
Packing Specification：
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R2118-20
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Fibrous tissue RNA Extraction Kit(SpinColumn)
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20T
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CNY610
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R2118-50
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Fibrous tissue RNA Extraction Kit(SpinColumn)
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50T
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CNY1100
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R2118-200
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Fibrous tissue RNA Extraction Kit(SpinColumn)
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200T
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CNY3960
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For research use only. Not for use in medicine, clinical diagnosis, food, cosmetics or other applications.
Product Introduction:
The unique lysis buffer/β-mercaptoethanol rapidly lyses cells and inactivates cellular RNases. After adjusting the binding conditions 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.
Product Features:
1.The silica matrix membrane in the spin column is made of high-quality special adsorption membrane, with minimal differences in adsorption capacity between columns.
2.No toxic reagents such as phenol or chloroform are used, and no ethanol precipitation steps are required.
3.Fast and simple: Operation for a single sample can generally be completed within 30 minutes.
4.Multiple column washing steps ensure high purity: The typical OD260/OD280 ratio ranges from 1.9 to 2.0, with almost no DNA residue. Suitable for RT-PCR, Northern blot, and various other experiments.
Experimental Procedures:
Notes
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!
Before operation, add β-mercaptoethanol to Lysis Buffer RLT to a final concentration of 1% (e.g., add 10 μL of β-mercaptoethanol to 1 mL of RLT). This lysis buffer is best prepared fresh. The prepared RLT can be stored at 4°C for one month.
1.Thorough Homogenization (Extremely Important; Otherwise, Yield Will Be Severely Reduced)
a.Mechanical Homogenization (Preferred and Highly Recommended for Highest Yield and Most Stable Results): Take approximately 10-20 mg (<30 mg) of fresh tissue, add 300 μL of Lysis Buffer RLT, and thoroughly homogenize the tissue cells using a rotor-stator homogenizer (e.g., TissueRupter) or a bead mill (e.g., TissueLyser) according to the manufacturer's instructions.
b.Liquid Nitrogen Grinding + Homogenization: Grind the tissue into fine powder in liquid nitrogen, transfer an appropriate amount (10-20 mg, <30 mg) of the fine tissue powder to a 1.5 mL centrifuge tube containing 300 μL of Lysis Buffer RLT, and vortex vigorously by hand for 20 seconds to fully lyse the sample. Aspirate and dispense the lysate 10 times using a disposable 1 mL syringe with a blunt needle (0.9 mm needle) or until a satisfactory homogenate is obtained (or homogenize electrically for 30 seconds) to shear DNA, reduce viscosity, and improve yield. Transfer the homogenate to a new centrifuge tube.
c.Mortar Grinding + Homogenization: Place an appropriate amount (10-20 mg, <30 mg) of fine tissue powder into a small mortar, quickly add 300 μL of Lysis Buffer RLT, and grind thoroughly into a homogenate at room temperature. Transfer the homogenate to a new centrifuge tube. Note: If significant homogenate adheres to the mortar and is lost, appropriately increase the initial tissue amount and Lysis Buffer RLT volume proportionally. Additionally, grind the tissue into fine powder in liquid nitrogen first, then add Lysis Buffer RLT immediately after the liquid nitrogen has just evaporated and grind thoroughly to improve homogenization efficiency.
2.Add 590 μL of RNase-free H2O to the homogenate, then add 10 μL of Proteinase K, and pipette to mix well.
3.Incubate in a 55°C water bath for 10 minutes.
4.Centrifuge at 13,000 rpm at room temperature for 5 minutes. A small pellet of tissue debris will form, and a small amount of floating matter may be visible on top of the supernatant.
5.Transfer the supernatant to a new 1.5 mL centrifuge tube. Note: Do not transfer the pellet, and ensure the pipette tip is placed below the floating matter on the supernatant. The floating matter may adhere to the outer wall of the pipette tip; do not transfer it to the centrifuge tube.
6.Accurately estimate the volume of the lysate (supernatant), add 0.5 volumes of absolute ethanol (generally 450 μL). Precipitation may occur at this time, but it will not affect the extraction process. Immediately pipette to mix well; do not centrifuge.
7.Immediately transfer the mixed solution (less than 700 μL each time, can be added in two portions) to the same SpinRA column (place the spin column into a collection tube), centrifuge at 13,000 rpm for 60 seconds, and discard the waste liquid.
8.Add 700 μL of Deproteinization Buffer RW1, incubate at room temperature for 30 seconds, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid. If DNA residue is significant, incubate at room temperature for 5 minutes after adding RW1 before centrifugation.
9.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.
10.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.
11.Remove the SpinRA column, place it into a new RNase-free centrifuge tube. According to the expected RNA yield, add 30-50 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.
Optional: To obtain higher RNA concentration, add the first eluate back to the spin column and repeat Step 13. The RNA concentration can be appropriately increased by double elution. If the expected RNA yield is >30 μg, add 30-50 μL of RNase-free water and repeat Step 13, then combine the two eluates to increase yield by 15–30%, but the concentration will be slightly lower. Users can choose according to their needs.
