Products

Product Introduction：
Based on the rapid phenol- and chloroform-free RNA extraction technology launched by our company, we have successfully developed genomic DNA removal column technology to ensure effective elimination of gDNA residues. The obtained RNA does not require DNase digestion and can be directly used in experiments such as PCR and quantitative real-time PCR (qPCR). The unique lysis buffer rapidly lyses cells and inactivates cellular RNases. The lysis mixture is then passed through a genomic DNA removal column, where genomic DNA is retained while RNA passes through the filter. After adjusting the binding conditions of the filtered RNA with ethanol, the RNA selectively binds to the silica matrix membrane inside the spin column under high chaotropic salt conditions. Through a series of rapid washing-centrifugation steps, deproteinization buffer and washing buffer remove impurities such as cellular metabolites and proteins. Finally, pure RNA is eluted from the silica matrix membrane using low-salt RNase-free H2O.

Product Features：
1.The silica matrix membranes in the spin columns are all high-quality specially modified membranes, ensuring minimal variation in adsorption capacity between columns.
2.No toxic reagents such as phenol or chloroform are required, and ethanol precipitation steps are eliminated.
3.Fast and simple operation; a single sample can be processed within 30 minutes.
4.Complete removal of DNA residues by DNase I digestion, enabling direct use in experiments such as PCR and qPCR.
5.Multiple column washing steps ensure high purity, with a typical OD260/OD280 ratio of 2.1-2.2 (the ratio of 100% pure RNA is generally around 2.2).

Application Scope：
Suitable for rapid extraction of total RNA from animal cells and easily lysed animal tissues. Complete removal of DNA residues by DNase I digestion allows the RNA to be directly used in PCR and qPCR experiments.

Operation Steps：
Note: Before first use, add the specified volume of anhydrous ethanol to the 70% ethanol bottle and Washing Buffer RW bottle, mix thoroughly. Immediately mark the corresponding checkbox after adding ethanol to avoid repeated addition!

Pre-Experiment Preparation
Take a new 1.5 mL centrifuge tube, add 45 μL DNase I Buffer and 5 μL RNase-free DNase I, gently pipette to mix and prepare DNase I working solution (scale up the volume proportionally when processing multiple columns). Store on ice for later use.

1.Cultured Cells
a.Collect <107 suspension cells into a 1.5 mL centrifuge tube. For adherent cells, lyse directly in the culture plate; for cells cultured in flasks, digest with trypsin first, then pipette to collect.
b.Centrifuge at 13,000 rpm for 10 seconds (or 300×g for 5 minutes) to pellet the cells. Completely aspirate and discard the supernatant, leaving the cell pellet. Note that 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 (<5×10⁶ cells) or 600 μL (5×106-1×107 cells) of Lysis Buffer RLT Plus, pipette to mix, then vigorously vortex by hand for 20 seconds to fully lyse the cells.
d.Homogenization: (Not required for very small cell amounts <1×10⁵; vortex for 1 minute to homogenize). Vigorously pipette the lysate more than 10 times with a disposable 1 mL syringe fitted with a blunt 0.9 mm needle (or homogenize electrically for 30 seconds) to shear DNA, reduce viscosity, prevent column clogging, and improve yield.
e.Centrifuge the homogenized lysate at 13,000 rpm for 3 minutes to pellet any difficult-to-lyse debris or insoluble matter. Transfer the entire supernatant to a new centrifuge tube. Add anhydrous ethanol equal to half the volume of the supernatant (0.5 volumes). Precipitation may form at this step but will not affect the extraction process. Immediately pipette to mix; do not centrifuge.
f.Proceed directly to Step 3 in the "General Operation Steps" below.

General Operation Steps
2.Animal Tissues (e.g., Mouse Liver, Brain)
a.Electrical Homogenization: Quickly cut fresh tissue into small pieces with a scalpel. Add 350 μL (<20 mg tissue) or 600 μL (20-30 mg tissue) of Lysis Buffer RLT Plus, 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 powder (20 mg/30 mg) into a 1.5 mL centrifuge tube containing 350 μL/600 μL of Lysis Buffer RLT Plus. Vigorously vortex by hand for 20 seconds to fully lyse the tissue. Vigorously pipette the lysate 10 times with a disposable 1 mL syringe fitted with a blunt 0.9 mm needle (or homogenize electrically for 30 seconds) to shear DNA, reduce viscosity, prevent column clogging, and improve yield.
c.Centrifuge the homogenized lysate at 13,000 rpm for 3 minutes to pellet any difficult-to-lyse debris or insoluble matter. Transfer the entire supernatant to a new centrifuge tube.
d.Add anhydrous ethanol equal to half the volume of the supernatant (0.5 volumes). Precipitation may form at this step but will not affect the extraction process. Immediately pipette to mix; do not centrifuge.
e.Proceed directly to Step 3 in the "General Operation Steps" below.

3.Immediately load the mixture (≤700 μL per load; load in two batches if the volume exceeds 700 μL) into a SpinRA RNA Spin Column (place the column in a collection tube). Centrifuge at 13,000 rpm for 30 seconds, discard the waste liquid, and ensure all liquid has passed through the filter after centrifugation.

4.Add 700 μL of Deproteinization Buffer RW1, incubate at room temperature for 1 minute, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

5.Add 50 μL of the prepared DNase I working solution to the center of the SpinRA RNA Spin Column. Incubate at room temperature (20-30℃) for 15 minutes. Note: Drop the working solution directly onto the center of the membrane; do not let it touch the O-ring or the inner wall of the centrifuge column.

6.Add 350 μL of Deproteinization Buffer RW1 to the SpinRA RNA Spin Column, centrifuge at 12,000 rpm for 30-60 seconds, discard the waste liquid, and place the spin column back into the collection tube.

7.Add 500 μL of Washing Buffer RW (please check if anhydrous 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 Washing Buffer RW.

8.Place the SpinRA RNA Spin Column back into an empty collection tube, centrifuge at 13,000 rpm for 2 minutes to thoroughly remove residual washing buffer, as ethanol residues in the washing buffer may inhibit downstream reactions.

9.Remove the SpinRA RNA Spin Column and place it into an RNase-free centrifuge tube. Add 30-50 μL of RNase-free water to the center of the adsorption membrane (preheating the water in a 70-90℃ water bath in advance can improve yield) based on the expected RNA yield. Incubate at room temperature for 1 minute, then centrifuge at 12,000 rpm for 1 minute.

10.If the expected RNA yield is >30 μg, repeat Step 7 with 30-50 μL of RNase-free water and combine the two eluates. Alternatively, reuse the first eluate by adding it back to the spin column and repeating Step 7 (for higher RNA concentration).
Eluting twice yields RNA with higher concentration. Combining eluates from two separate elutions results in 15-30% higher RNA yield but lower concentration. Choose the method based on experimental needs.