Products

Micro Cell/Tissue RNA Extraction Kit with DNase I(SpinColumn)

Packing Specification：

Product Introduction：
The unique lysis buffer 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 2.1 to 2.2 (The ratio of 100% pure RNA is generally around 2.2), with almost no DNA residue. Suitable for RT-PCR, Northern blot, and various other experiments.

Application Scope：
Suitable for the rapid extraction of total RNA from various micro-scale cells/tissues.

Experimental Procedures：
Note:Before first use, add the indicated amount of absolute ethanol to the Wash Buffer RW bottle and 70% Ethanol bottle, mix thoroughly, and mark with a check in the box immediately after adding to avoid repeated addition!
1.Cultured Cells
a.Collect <5×10⁵ suspension cells into a 1.5 mL centrifuge tube. For adherent cells: Lyse directly if cultured in well plates; if cultured in flasks, digest with trypsin first, pipette to detach, and then collect.
b.Centrifuge at 13,000 rpm for 10 seconds (or 300 g for 5 minutes) to pellet the cells. Aspirate the supernatant completely, leaving the cell pellet. Note: Incomplete removal of the supernatant will dilute the lysis buffer, leading to reduced yield and purity.
c.Flick the tube wall to fully resuspend the cell pellet. Add 350 μL of Lysis Buffer RLT (<5×10⁵ cells) and 4 μL of Poly Carrier, pipette to mix well, then vortex vigorously by hand for 20 seconds to fully lyse.
d.Homogenize the lysate by drawing and expelling 5-10 times with a disposable 1 mL syringe equipped with a 0.9 mm blunt needle until a satisfactory homogenate is obtained (or homogenize electrically for 30 seconds). This can shear DNA, reduce viscosity, and improve yield.
e.Proceed to Step 3 of the experimental procedures.

2.Animal Tissues (e.g., Mouse Liver, Brain)
a.Electrical Homogenization:Quickly cut fresh tissue into small pieces with a scalpel. Add 350 μL of Lysis Buffer RLT (<5 mg tissue) and 4 μL of Poly Carrier, then homogenize thoroughly electrically for 20-40 seconds.
b.Liquid Nitrogen Grinding + Homogenization:Grind the tissue into fine powder in liquid nitrogen. Transfer an appropriate amount of tissue fine powder (less than 10 mg) to a 1.5 mL centrifuge tube containing 350 μL of Tissue Lysis Buffer RLT, vortex vigorously by hand for 20 seconds to fully lyse. Homogenize the lysate by drawing and expelling 10 times with a disposable 1 mL syringe equipped with a 0.9 mm blunt needle until a satisfactory homogenate is obtained (or homogenize electrically for 30 seconds). This can shear DNA, reduce viscosity, and improve yield.
c.Centrifuge the homogenized lysate at 13,000 rpm for 2 minutes to pellet any hard-to-lyse debris or insoluble substances. Carefully transfer the lysate supernatant to a new centrifuge tube.
d.Proceed to Step 3 of the experimental procedures.

Experimental Procedures (Continued)
3.Accurately estimate the volume of the lysate (supernatant), add an equal volume of 70% ethanol (please check if absolute ethanol has been added first!). Precipitation may occur at this time, but it will not affect the extraction process. Immediately pipette to mix well; do not centrifuge.

4.Immediately transfer the mixture to a micro spin column (RA), place the spin column into a collection tube, centrifuge at 13,000 rpm for 60 seconds, and discard the waste liquid.

5.Add 350 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.

6.Preparation of DNase I Working Solution: Take 20 μL of DNase I Buffer and 2 μL of RNase-Free DNase I into a centrifuge tube, and gently pipette to mix well to prepare the working solution (scale up the volume proportionally when processing multiple columns).

7.Add 22 μL of the DNase I working solution to the center of the spin column (RA). Incubate at room temperature (20-30°C) for 15 minutes. Note: Add the working solution directly to the center of the membrane; do not let it drip onto the O-ring or the inner wall of the centrifuge column.

8.Add 350 μL of Deproteinization Buffer RW1, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

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 spin column (RA) 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 spin column (RA), place it into a new RNase-free centrifuge tube. According to the expected RNA yield, add 20-30 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.