Products

Berry RNA Extraction Kit(SpinColumn)

Packing Specification：

Product Introduction：
Berry fruits (e.g., grapes, kiwifruits, figs, watermelons, tomatoes, strawberries, oranges, mulberries, pomegranates) contain complex organic substances such as polysaccharides, polyphenols, gums, flavonoids, and moisture, making it difficult to efficiently and completely isolate and extract RNA. Lysis Buffer PLB has been optimized and improved multiple times to address these issues, successfully solving this problem. Berry fruits are homogenized and lysed in a lysis buffer containing high concentrations of guanidinium salt, and RNA is released into the lysis buffer. Due to the denaturants in the lysis buffer, endogenous or exogenous RNases are denatured and inactivated, protecting RNA. Insoluble impurities are removed from the lysis buffer by centrifugation. The resulting supernatant is filtered through a gDNA filter column to remove genomic DNA. Ethanol is added to the filtrate to adjust binding conditions, and the mixture is transferred to a spin column for filtration. RNA is adsorbed onto the membrane of the column, while proteins and impurities are not adsorbed and are removed. The column is washed with Deproteinization Buffer RW1 to remove proteins and other impurities, and with Wash Buffer RW to remove salts. Finally, RNA is eluted with RNase-Free H2O.

Product Features：
1.Capable of extracting RNA from berries rich in complex organic substances such as polysaccharides, polyphenols, gums, flavonoids, and moisture.
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.No toxic reagents such as phenol or chloroform are used, and no ethanol precipitation steps are required.
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：
Note:Before first use, add the indicated amount of absolute ethanol to the Wash Buffer RW bottle!
1.Transfer 900 μL of Lysis Buffer PLB to a 2 mL centrifuge tube (if PLB has precipitated, dissolve it again in a 65°C water bath first). Add β-mercaptoethanol to a final concentration of 10% (100 μL of β-mercaptoethanol per 900 μL of PLB). Invert to mix well and preheat in a 65°C water bath. Scale up proportionally for multiple samples.
2.Direct Grinding Method (Recommended for laboratories without liquid nitrogen or soft, easy-to-grind plant samples)
a.Take 500 mg of berry fruit (300 mg for low-moisture pulp samples; more for high-moisture samples such as grapes and watermelons) and place it into a mortar. Add 1 mL of PLB (with 100 μL of 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 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, quickly 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 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.
d.Proceed directly to Step 4 of the experimental procedures.

3.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 the fine powder to a 2 mL centrifuge tube containing 1 mL of Lysis Buffer PLB (with 10% β-mercaptoethanol), fill the entire centrifuge tube, then invert to mix well. Scrape and fill the centrifuge tube again to improve RNA yield. Immediately vortex vigorously for 30-60 seconds.
c.Quickly 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 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 invert to mix well; do not centrifuge.
f.Proceed directly to Step 4 of the experimental procedures.

Experimental Procedures (Continued)

4.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.

5.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). Add 250 μL 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.

6.Immediately transfer the mixture 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.
7.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.

8.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.

9.Add another 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.

10.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.

11.Carefully 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 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 11, then combine the two eluates. Alternatively, add the first eluate back to the spin column and repeat Step 11 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.