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Officinal Plant RNA Extraction Kit(SpinColumn)

Packing Specification：

Product Introduction：
Medicinal plants are rich in complex secondary metabolites such as polysaccharides, polyphenols, pigments, starches, and terpenoids, which interfere with the extraction of total RNA. Conventional extraction methods often fail to obtain total RNA or result in severe RNA degradation. These complex secondary metabolites can bind to DNA to form complexes, trapping DNA in viscous colloids that are difficult to dissolve, and may even cause browning to varying degrees.
Under the condition of high-concentration salt ion reagents (such as guanidine hydrochloride or guanidine isothiocyanate), this kit can adsorb nucleic acids through physical and chemical interactions such as hydrogen bonding and electrostatic forces, while proteins and other impurities are not adsorbed and are removed. The filter membrane with adsorbed nucleic acids is washed to remove residual proteins and salts. Finally, nucleic acids adsorbed on the filter membrane can be eluted with a low-salt buffer (such as Buffer TE) or water. The obtained nucleic acids have high purity and can be directly used in various downstream experiments. The DNA filter column uses a special adsorption membrane that can efficiently and selectively adsorb genomic DNA without adsorbing RNA.

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 25 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 reverse transcription PCR (RT-PCR), quantitative real-time PCR, and other experiments.
4.Wide applicability: Can extract RNA from hundreds of samples that fail with other domestic and foreign kits, including complex Chinese medicinal materials (e.g., Dendrobium, Salvia miltiorrhiza, Saussurea involucrata, Ginseng), complex starchy seeds (e.g., rice, wheat, corn seeds), complex fruits (e.g., grape, blueberry, strawberry, watermelon fruits), complex stress-tolerant plants (e.g., Ilex chinensis, pine needles, sea-buckthorn, Populus euphratica), complex flowers (e.g., rose, Chinese rose, plum, peony), and complex polysaccharide-rich plants (e.g., laver, cactus, aloe, rice seeds).
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 and purification of high-quality RNA from medicinal plant tissues rich in complex secondary metabolites such as polysaccharides, polyphenols, pigments, starches, and terpenoids. Examples include RNA extraction from plants such as Rheum officinale, Dendrobium, Salvia miltiorrhiza, Saussurea involucrata, and Ginseng. The genomic DNA removal column effectively eliminates gDNA residue without the need for DNase digestion, resulting in RNA with high purity and no residual DNA or impurities. It is suitable for downstream experiments requiring high purity, such as real-time RT-PCR and RNA-Seq.

Experimental Procedures：
Pre-Experiment Preparation
1.Before first use, add the indicated amount of absolute ethanol to the Wash Buffer RW bottle!
2. Transfer 1 mL of Lysis Buffer PLB to a centrifuge tube (if PLB has precipitated, dissolve it again in a 65°C water bath first). Add 5% β-mercaptoethanol to Lysis Buffer PLB (50 μL of β-mercaptoethanol per 1 mL of PLB). Invert to mix well and preheat in a 65°C water bath. Scale up proportionally for multiple samples.
1.Direct Grinding Method (Recommended for laboratories without liquid nitrogen or soft, easy-to-grind plant samples)
a.Weigh 100-200 mg of fresh or frozen plant tissue (100-150 mg for low-moisture samples such as leaves and seeds; more for high-moisture samples such as strawberry and watermelon fruits). Cut into small pieces quickly and place into a mortar. Add 1 mL of PLB (with β-mercaptoethanol) and grind thoroughly into a homogenate at room temperature. Note: Grind quickly to ensure immediate and full contact between the tissue and Lysis Buffer PLB to inhibit RNase activity. β-mercaptoethanol is a key component of Lysis Buffer PLB; its final concentration can be increased to 10-20% if necessary. For particularly complex plants, try adding PVP40 to the lysis buffer to a final concentration of 2%.
b.Transfer the lysate to a centrifuge tube, vortex vigorously for 15 seconds immediately, place back in the 65°C water bath for 5 minutes, and occasionally invert 1-2 times to assist lysis. Centrifuge at 13,000 rpm for 5 minutes to pellet undigested debris.
c.Transfer the lysate supernatant (up to 750 μL each time) to a filter column, centrifuge for 1 minute, and retain the filtrate. If the supernatant cannot be filtered at once, split it into two portions.
d.Transfer 500 μL of the filtrate to a new centrifuge tube (more filtrate can be taken if it does not exceed the capacity of the genomic DNA removal column to increase yield). Add 250 μL of absolute ethanol (0.5 volumes of the filtrate). 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.
e.Proceed directly to Step 3 of the experimental procedures.

2.Liquid Nitrogen Grinding Method (Widely applicable; recommended for extracting complex, hard-to-break, and easily degradable samples)
a.Grind fresh or -70°C frozen samples into fine powder in liquid nitrogen.
b.Transfer 100-200 mg of fine powder (100-150 mg for low-moisture samples such as leaves and seeds; more for high-moisture samples such as strawberry and watermelon fruits) to the preheated Lysis Buffer PLB (with β-mercaptoethanol) centrifuge tube. 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.Place back in the 65°C water bath for 5 minutes, occasionally inverting 1-2 times to assist lysis.
d.Centrifuge the lysate at 13,000 rpm for 5 minutes to pellet undigested debris.
e.Transfer the lysate supernatant (up to 750 μL each time) to a filter column, centrifuge for 1 minute, and retain the filtrate. If the supernatant cannot be filtered at once, split it into two portions.
f.Transfer 500 μL of the filtrate to a new centrifuge tube (more filtrate can be taken if it does not exceed the capacity of the genomic DNA removal column to increase yield). Add 250 μL of absolute ethanol (0.5 volumes of the filtrate). 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.
g.Proceed directly to Step 3 of the experimental procedures.

Experimental Procedures (Continued)
3.Transfer the mixed solution (less than 750 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 1 minute, 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.

4.Place the genomic DNA removal column into a clean 2 mL centrifuge tube. Add 500 μL of Lysis Buffer RLT Plus to the genomic DNA removal column, centrifuge at 13,000 rpm for 30 seconds, 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.
Immediately transfer the mixed solution (less than 750 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 1 minute, 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.

5.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.

6.Add 700 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.

7.Add another 500 μL of Wash Buffer RW (please check if absolute ethanol has been added first!), centrifuge at 13,000 rpm for 3 minutes to remove as much wash buffer as possible, avoiding residual ethanol in the wash buffer that may inhibit downstream reactions.

8.Carefully remove the SpinRA column (do not let the SpinRA column touch the waste liquid in the collection tube), place it into a new RNase-free centrifuge tube. According to the expected RNA yield, add 30-50 μL of RNase-free H₂O to the center of the adsorption membrane (heating the water in a 70-90°C water bath 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 H₂O and repeat Step 9, then combine the two eluates. Alternatively, add the first eluate back to the spin column and repeat Step 9 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.