Complex Plant RNA Extraction Kit(SpinColumn)
Packing Specification：
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R2117-50
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Complex Plant RNA Extraction Kit(SpinColumn)
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50T
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CNY1560
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R2117-200
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Complex Plant RNA Extraction Kit(SpinColumn)
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200T
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CNY5616
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For research use only. Not for use in medicine, clinical diagnosis, food, cosmetics or other applications.
Product Introduction:
Based on our exclusive spin column-based rapid RNA extraction technology without phenol or chloroform, we have successfully developed genomic DNA removal column technology that 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, polyphenols, and secondary metabolites are removed by centrifugation. The lysis mixture is adjusted with ethanol to enable RNA binding and adsorption to the genomic DNA removal column. RNA is then selectively eluted and filtered, while residual DNA adsorbed on the genomic DNA removal column cannot be eluted and is discarded along with the column, thus removing 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 25 minutes.
3.Genomic DNA removal column technology effectively eliminates gDNA residue. The obtained RNA generally does not require DNase digestion and is suitable for RT-PCR, quantitative real-time PCR, and other experiments.
4.Wide applicability: Can extract RNA from hundreds of complex samples that fail to be extracted by many domestic and foreign kits, including complex Chinese herbal medicines (Dendrobium, Salvia miltiorrhiza, Saussurea involucrata, Ginseng), complex starchy seeds (rice, wheat, corn seeds), complex fruits (grape, blueberry, strawberry, watermelon fruits), complex stress-tolerant plants (holly, pine needles, sea-buckthorn, Populus euphratica), complex flowers (rose, peony), and complex polysaccharide-containing plants (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.
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!
Transfer 1 mL of Lysis Buffer CLB to a centrifuge tube (if CLB has precipitated, dissolve it again in a 65°C water bath first). Add 5% β-mercaptoethanol to Lysis Buffer CLB (50 μL of β-mercaptoethanol per 1 mL of CLB). 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 CLB (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 CLB to inhibit RNase activity. β-mercaptoethanol is a key component of Lysis Buffer CLB; its final concentration can be increased to 10-20% if necessary. For particularly complex plants, PVP40 can be added 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 a short time (5-10 minutes), and occasionally invert 1-2 times to assist lysis. Centrifuge at 13,000 rpm for 10 minutes to pellet undigested debris.
c.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.
d.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 CLB (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 a short time (5-10 minutes), occasionally inverting 1-2 times to assist lysis.
d.Centrifuge the lysate at 13,000 rpm for 10 minutes to pellet undigested debris.
e.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.
f.Proceed directly to Step 3 of the experimental procedures.
Experimental Procedures (Continued)
3.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.
4.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 provided with 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 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.
5.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.
6.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.
7.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.
8.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.
9.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 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.
