Products

Plant microRNA Extraction Kit(SpinColumn)

Packing Specification：

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

Product Introduction
The unique lysis buffer rapidly lyses cells and inactivates cellular RNases. The plant RNA auxiliary agent PLANTaid helps bind polysaccharides and polyphenols, which are then removed by centrifugation. The lysis mixture is passed through a genomic DNA removal column, where genomic DNA is retained while RNA (including microRNA) is selectively filtered. 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 with special wash solutions, impurities such as cellular metabolites and proteins are removed. Finally, pure RNA (including microRNA) is eluted from the silica matrix membrane with low-salt RNase-free H2O.
Using separate extraction steps, the enriched microRNA fraction and total RNA (>200 nt) can also be purified individually to obtain isolated and enriched microRNA and RNA.

Product Features:
1.No toxic reagents such as phenol or chloroform are used, and no ethanol precipitation steps are required.
2.Fast and simple: Operation for a single sample can generally be completed within 30 minutes.
3.The exclusive plant RNA auxiliary agent can effectively bind polysaccharides and polyphenols, improving removal efficiency.
4.Multiple column washing steps ensure high purity: The typical OD260/OD280 ratio ranges from 2.0 to 2.2. Suitable for RT-PCR, Northern blot, and various other experiments.

Application Scope:
Suitable for the rapid extraction of plant microRNA, or separate extraction of microRNA and total RNA.

Experimental Procedures：
Experimental Notes
Before first use, add the indicated amount of absolute ethanol to the Wash Solution 1 and Wash Solution 2/3 bottles!
For plant tissues rich in RNases or polyphenols: Add β-mercaptoethanol to Lysis Buffer RLT Plus to a final concentration of 1% before operation (e.g., add 10 μL of β-mercaptoethanol to 1 mL of RLT Plus). This lysis buffer is best prepared fresh before use. The prepared RLT Plus can be stored at 4°C for one month.
1.Direct Grinding Method (Recommended for extracting simple plant samples; liquid nitrogen grinding can also be used for simple samples)
a.Weigh 100 mg of fresh plant tissue, cut into small pieces quickly, and place into a mortar (frozen or liquid nitrogen-preserved samples can be weighed directly and 100 mg placed into a mortar). Add 10 volumes (1 mL) of RLT Plus and 1 volume (100 μL) of PLANTaid, grind thoroughly into a homogenate at room temperature. Note: Grind quickly to ensure immediate and full contact between the tissue and Lysis Buffer RLT Plus to inhibit RNase activity.
Note: PLANTaid is an indispensable component for extracting difficult samples rich in polysaccharides, polyphenols, secondary metabolites, and pigments.
b.Transfer the lysate to a centrifuge tube, vortex vigorously for 15 seconds, centrifuge at 13,000 rpm for 5-10 minutes to pellet undigested debris and PLANTaid bound to polysaccharides and polyphenols.
c.Take 480 μL of the lysate supernatant (more supernatant can be taken if it does not exceed the capacity of the genomic DNA removal column to increase yield; if there is excessive residual genomic DNA, appropriately reduce the amount of supernatant taken) and load it onto the DNA removal column (place the column into a collection tube).
d.Proceed directly to Step 3 of the experimental procedures.
2.Liquid Nitrogen Grinding Method (Recommended for extracting complex, easily degradable samples)
a.Transfer 500 μL of Lysis Buffer RLT Plus to a 1.5 mL centrifuge tube, add 50 μL of PLANTaid, and mix well for later use.
b.Grind an appropriate amount of plant tissue into fine powder in liquid nitrogen, transfer 50 mg of the fine powder to the aforementioned centrifuge tube containing RLT Plus and PLANTaid, and vortex vigorously by hand for 20 seconds to fully lyse.
c.Pipette to mix well to assist lysis or vortex vigorously until a satisfactory homogenate is obtained (or homogenize electrically for 30 seconds). This can shear DNA, reduce viscosity, and improve yield.
d.Centrifuge the lysate at 13,000 rpm for 5-10 minutes to pellet undigested debris and PLANTaid bound to polysaccharides and polyphenols.
e.Take the lysate supernatant (more supernatant can be taken if it does not exceed the capacity of the genomic DNA removal column to increase yield) and load it onto the DNA removal column (place the column into a collection tube).
f.Proceed directly to Step 3 of the experimental procedures.
Note: Users can double the processing volume as needed for the liquid nitrogen grinding method to improve yield (i.e., use 1 mL of Lysis Buffer RLT Plus, 100 μL of PLANTaid, and 100 mg of sample).

Experimental Procedures (Continued)
3.Centrifuge immediately at 13,000 rpm for 60 seconds and collect the filtrate (RNA is in the filtrate).
4.Use a micropipette to accurately estimate the volume of the filtrate (approximately 480 μL; subtract the volume lost during filtration), add 1.25 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 700 μL each time, can be added in two portions) to a Spin RA column (place the spin column into a collection tube), centrifuge at 13,000 rpm for 30 seconds, 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 Wash Solution 1 (please check if absolute ethanol has been added first!), centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.
7.Add 500 μL of Wash Solution 2/3 (please check if absolute 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 Wash Solution 2/3.
8.Place the Spin RA 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 Spin RA 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, incubate at room temperature for 1 minute, and centrifuge at 12,000 rpm for 1 minute.
Re-adding the eluate to the spin column and repeating the elution step can increase the yield by approximately 10-15%.
The obtained RNA/microRNA can be used immediately for downstream reactions or stored at low temperature as soon as possible.

Appendix 1: microRNA Enrichment Method (Separate Extraction of microRNA and Total RNA Depleted of microRNA)
For plant tissues rich in RNases or polyphenols: Add β-mercaptoethanol to Lysis Buffer RLT Plus to a final concentration of 1% before operation (e.g., add 10 μL of β-mercaptoethanol to 1 mL of RLT Plus). This lysis buffer is best prepared fresh before use. The prepared RLT Plus can be stored at 4°C for one month.
1.Direct Grinding Method (Recommended for extracting simple plant samples; liquid nitrogen grinding can also be used for simple samples)
a.Weigh 100 mg of fresh plant tissue, cut into small pieces quickly, and place into a mortar (frozen or liquid nitrogen-preserved samples can be weighed directly and 100 mg placed into a mortar). Add 10 volumes (1 mL) of RLT Plus and 1 volume (100 μL) of PLANTaid, grind thoroughly into a homogenate at room temperature.Note: Grind quickly to ensure immediate and full contact between the tissue and Lysis Buffer RLT to inhibit RNase activity.
Note: PLANTaid is an indispensable component for extracting difficult samples rich in polysaccharides, polyphenols, secondary metabolites, and pigments.
b.Transfer the lysate to a centrifuge tube, vortex vigorously for 15 seconds, centrifuge at 13,000 rpm for 5-10 minutes to pellet undigested debris and PLANTaid bound to polysaccharides and polyphenols.
c.Take 480 μL of the lysate supernatant (more supernatant can be taken if it does not exceed the capacity of the genomic DNA removal column to increase yield; if there is excessive residual genomic DNA, appropriately reduce the amount of supernatant taken) and transfer to a new centrifuge tube. 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 3 of the enrichment method.

2.Liquid Nitrogen Grinding Method (Recommended for extracting complex, easily degradable samples)
a.Transfer 500 μL of Lysis Buffer RLT Plus to a 1.5 mL centrifuge tube, add 50 μL of PLANTaid, and mix well for later use.
b.Grind an appropriate amount of plant tissue into fine powder in liquid nitrogen, transfer 50 mg of the fine powder to the aforementioned centrifuge tube containing RLT Plus and PLANTaid, and vortex vigorously by hand for 20 seconds to fully lyse.
c.Pipette to mix well to assist lysis or vortex vigorously until a satisfactory homogenate is obtained (or homogenize electrically for 30 seconds). This can shear DNA, reduce viscosity, and improve yield.
d.Centrifuge the lysate at 13,000 rpm for 5-10 minutes to pellet undigested debris and PLANTaid bound to polysaccharides and polyphenols.
e.Take the lysate supernatant (more supernatant can be taken if it does not exceed the capacity of the genomic DNA removal column to increase yield) and transfer to a new centrifuge tube. 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.
f.Proceed directly to Step 3 of the enrichment method.
Note: Users can double the processing volume as needed for the liquid nitrogen grinding method to improve yield (i.e., use 1 mL of Lysis Buffer RLT Plus, 100 μL of PLANTaid, and 100 mg of sample).

Enrichment Method Procedures
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 retain the filtrate (microRNA is in the filtrate).
At this point, the filtrate contains microRNA, and the genomic DNA removal column retains total RNA depleted of microRNA (excluding microRNA). If needed, perform washing and elution according to Steps 6-10 of the standard operation procedures above to recover the total RNA depleted of microRNA.
4.Use a micropipette to accurately estimate the volume of the filtrate, add 0.65 volumes of room-temperature absolute ethanol, vortex or pipette to mix thoroughly; do not centrifuge.
5.Immediately transfer the mixed solution (less than 700 μL each time, can be added in two portions) to a Spin RA 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.Perform washing and elution according to Steps 6-10 of the standard operation procedures above to obtain enriched microRNA.
Note: Different methods can be selected for different experiments. For example, Northern Blot or expression microarray analysis can use total RNA including microRNA. Enriched microRNA removes larger mRNA and rRNA fragments, which may reduce amplification background in some downstream experiments. For high background or excessive non-specific amplification, try using enriched microRNA.

Appendix 2: 
On-Column DNase Digestion (For details, refer to the R2127 DNase I On-Column Digestion Kit manual)
1.Perform the operation procedures listed above until completing Step 5.
2.Prepare the DNase I working solution: Take 45 μL of DNase I Buffer and 5 μL of RNase-free DNase I into a centrifuge tube, and gently pipette to mix well (scale up proportionally when processing multiple columns).
3.Add 350 μL of Wash Solution 1 to the Spin RA column, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and place the spin column back into the collection tube.
4.Add 50 μL of the DNase I working solution to the center of the Spin RA column. Incubate at room temperature (20°C-30°C) for 15 minutes. Note: Add the working solution directly to the center of the membrane to ensure full infiltration and contact with the membrane. Do not allow the working solution to adhere to the O-ring or the inner wall of the centrifuge column, as this will prevent full contact with the membrane.
5.Add 350 μL of Wash Solution 1 to the Spin RA column, centrifuge at 12,000 rpm for 30-60 seconds, discard the waste liquid, and place the spin column back into the collection tube.
6.Proceed to Step 7 of the operation procedures to complete the subsequent steps.

Appendix 3:
Operation Procedures for the Plant microRNA Extraction Kit Using Lysis Buffer CLB
Before first use, add the indicated amount of absolute ethanol to the Wash Solution 1 and Wash Solution 2/3 bottles!
1.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 the 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.
2.Grind fresh or -70°C frozen samples into fine powder in liquid nitrogen. Transfer 100-150 mg of fine powder (100 mg for low-moisture samples such as seeds and leaves; more for high-moisture samples such as watermelons) 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. Place back in the 65°C water bath for a short time (5 minutes), occasionally inverting 1-2 times to assist lysis.
β-mercaptoethanol is a key component of Lysis Buffer CLB; its final concentration can be increased to 10-20% if necessary.
3.After vortexing to mix well, centrifuge at 13,000 rpm for 10 minutes at room temperature.
4.Take the lysate supernatant (more supernatant can be taken if it does not exceed the capacity of the genomic DNA removal column to increase yield) and transfer to a new centrifuge tube. If there is floating matter on the surface of the supernatant, use a pipette tip to move it aside and aspirate the liquid below.
5.Proceed directly to Step 3 of the operation procedures to complete the subsequent steps.