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Osseous Tissue RNA Extraction Kit(SpinColumn)

Packing Specification：

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

Product Introduction：
Osseous tissue is hard, has low osteocyte density, and its peripheral matrix contains a large amount of mucin (proteoglycans) that is difficult to separate from RNA, making high-quality extraction impossible with the traditional Trizol method. This kit uses a unique phenol/chloroform-free lysis buffer formula and adds multiple components to remove proteoglycans from osseous tissue. Meanwhile, the unique genomic DNA removal column technology can effectively eliminate gDNA residue. The obtained RNA generally does not require DNase digestion and can be used in reverse transcription PCR (RT-PCR), quantitative real-time PCR, and other experiments.

The unique lysis buffer rapidly lyses cells and inactivates cellular RNases. Polysaccharides and secondary metabolites are removed by centrifugation. The lysis mixture is then adjusted with ethanol to bind RNA to the genomic DNA removal column, and RNA is selectively eluted and filtered. Residual DNA adsorbed on the genomic DNA removal column cannot be eluted and is discarded along with the column to eliminate DNA. 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.

Product Features：
1.No toxic reagents such as phenol or chloroform are used, and no ethanol precipitation steps are required.
2.Simple and efficient: Operation for a single sample can generally be completed within 35 minutes.
3.Genomic DNA removal column technology effectively eliminates gDNA residue. The obtained RNA generally does not require DNase digestion and can be used in RT-PCR, quantitative real-time PCR, and other experiments.
4.Wide adaptability: Can extract RNA from various osseous tissues, including mineralized osseous tissue.
5.Multiple column washing steps ensure high purity: The typical OD260/OD280 ratio ranges from 1.9 to 2.2, with almost no DNA residue. Suitable for RT-PCR, Northern blot, next-generation sequencing, and various other experiments.

Application Scope：
Suitable for the rapid extraction of total RNA from osseous tissue cells. The genomic DNA removal column technology can effectively eliminate visually detectable gDNA residue by electrophoresis. The RNA can be used in RT-PCR, quantitative real-time PCR, and other experiments.

Experimental Procedures：
Notes
Before first use, add the indicated amount of absolute ethanol to the Wash Buffer RW bottle!
Transfer 1 mL of Lysis Buffer BL to a centrifuge tube (add β-mercaptoethanol to a final concentration of 10% in advance). Invert to mix well and preheat in a 65°C water bath. Scale up proportionally for multiple samples.

1.Liquid Nitrogen Grinding Method (Recommended for laboratories without ultra-low temperature tissue grinders)
a.Thoroughly crush the osseous tissue with bone forceps and place it into a mortar (the mortar should be dry-baked at 180°C for 2 hours). Alternatively, freeze-embed and section the osseous tissue sample in liquid nitrogen to crush it, then add liquid nitrogen and grind repeatedly into fine powder. Continuously replenish liquid nitrogen to keep it present after evaporation.
b.Transfer 50-100 mg of fine powder to the centrifuge tube containing 1 mL of preheated Lysis Buffer BL. Immediately vortex vigorously for 30-60 seconds or pipette to mix well for lysis until a satisfactory homogenate is obtained (or homogenize electrically for 30 seconds), which can shear DNA, reduce viscosity, and improve yield.
c.Proceed directly to Step 3 of the experimental procedures.

2.Ultra-Low Temperature High-Speed Tissue Grinder Method (Recommended for laboratories without liquid nitrogen)
a.Add 100 mg of osseous tissue to 1 mL of Lysis Buffer BL, and homogenize using an ultra-low temperature (-10°C) high-speed tissue homogenizer.
b.Proceed directly to Step 3 of the experimental procedures.
Experimental Procedures (Continued)
3.Place the mixed sample centrifuge tube into a 65°C water bath for 10-12 minutes, inverting 2-3 times during incubation to assist lysis.
4.Cool the lysate, centrifuge at high speed in a 4°C refrigerated centrifuge at 13,000 rpm for 5-10 minutes to pellet undigested debris.
5.Transfer the lysate supernatant to a new centrifuge tube (more supernatant can be taken if it does not exceed the capacity of the genomic DNA removal column to increase yield). Add absolute ethanol equal to 0.5 volumes of the supernatant. Precipitation may occur at this time, but it will not affect the extraction process. Immediately pipette to mix well; do not centrifuge. If there is floating matter on the surface of the supernatant, use a pipette tip to move it aside and aspirate the liquid below.
6.Transfer the mixed solution (less than 720 μL each time, can be added in two portions) to a genomic DNA removal column (place the column into a collection tube), centrifuge at 13,000 rpm for 2 minutes, and discard the waste liquid. Ensure that all liquid has passed through the membrane after centrifugation with no residue left on the membrane. If necessary, increase the centrifugal force and time.
7.Place the genomic DNA removal column into a clean 2 mL centrifuge tube (no need for RNase-free or DEPC treatment; a general clean new centrifuge tube is sufficient. The new clean collection tube accompanying the RNA spin column can also be used). Add 500 μL of Lysis Buffer RLT Plus to the genomic DNA removal column, centrifuge at 13,000 rpm for 1 minute, and collect the filtrate (RNA is in the filtrate). Use a micropipette to accurately estimate the volume of the filtrate (usually approximately 450-500 μL; subtract the volume lost during filtration), add 0.5 volumes of absolute ethanol. Precipitation may occur at this time, but it will not affect the extraction process. Immediately pipette to mix well; do not centrifuge.
8.Immediately transfer the mixed solution (less than 720 μL each time, can be added in two portions) to a SpinRA column (place the spin column into a collection tube), centrifuge at 13,000 rpm for 2 minutes, and discard the waste liquid. Ensure that all liquid has passed through the membrane after centrifugation with no residue left on the membrane. If necessary, increase the centrifugal force and time.
9.Add 700 μL of Deproteinization Buffer RW1, incubate at room temperature for 1 minute, centrifuge at 13,000 rpm for 30 seconds, and discard the waste liquid.
10.Add 500 μL of Wash Buffer RW (please check if absolute ethanol has been added first!), centrifuge at 13,000 rpm for 30 seconds, and discard the waste liquid. Repeat this step with another 500 μL of Wash Buffer RW.
11.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.
12.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.
If the expected RNA yield is >30 μg, add 30-50 μL of RNase-free water and repeat Step 12, then combine the two eluates. Alternatively, add the first eluate back to the spin column and repeat Step 12 for higher RNA concentration. 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.