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Serum/Plasma microRNA Extraction Kit(SpinColumn)

Packing Specification：

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

Product Introduction:
This kit uses a unique lysis buffer to rapidly lyse various animal serum and plasma samples, solubilize cellular contents, and inhibit nuclease activity, effectively preventing RNA degradation during extraction. Organic reagents are added for extraction to remove proteins and DNA. Under high-concentration ethanol conditions, RNA (including total RNA and small molecular RNAs such as microRNA, siRNA, snRNA, and other small RNAs <200 nt) binds to the special silica matrix membrane in the spin column. Through a series of rapid washing-centrifugation steps with special washing solutions, impurities such as cellular metabolites and proteins are further removed. Finally, pure RNA is eluted from the silica matrix membrane using a low-salt elution buffer.

Product Features:
1.The silica matrix membranes in the spin columns are all high-quality specially manufactured adsorption membranes, ensuring minimal variation in adsorption capacity between columns.
2.Few steps and simple operation: processing of a single sample can generally be completed within 30 minutes.
3.Multiple column washing steps ensure high purity, with a typical OD260/OD280 ratio of 2.1-2.2 (the ratio of 100% pure RNA is generally around 2.2).

Application Scope:
Suitable for purifying total RNA and small molecular RNAs (microRNA, siRNA, snRNA, and other small RNAs <200 nt) from animal and human serum and plasma.

Operation Steps:
Note: Before first use, add the specified volume of absolute ethanol to Wash Solution 1 (BR1) and Wash Solution 2 (BR2), mix thoroughly. Mark the corresponding box immediately after adding ethanol to avoid repeated additions!

1.Prepare serum or plasma samples in advance. Add 1 mL/2 mL/2.5 mL of Lysis Buffer to 200 μL/400 μL/500 μL of sample (serum/plasma). Vortex vigorously to mix, or pipette up and down several times to assist in lysis and mixing.
For samples with high contaminants, appropriately reduce the processing volume and make up the insufficient volume with RNase-free H2O.To obtain higher miRNA yield, the processing volume of serum/plasma samples can be increased to 250-1000 μL.The final volume ratio of Lysis Buffer to liquid sample must be 5:1. Additional Lysis Buffer can be purchased separately if needed.

2.Incubate the sample-containing lysis buffer at 75℃ for 5 minutes, then cool to room temperature and let stand for 3-5 minutes.

3.Add an equal volume of chloroform (200 μL/400 μL/500 μL) to the sample, vortex vigorously for 15 seconds, and incubate at room temperature (15–25°C) for 2-3 minutes. Thorough mixing is crucial for subsequent phase separation.

4.Centrifuge at 12,000 rpm at 4℃ for 15 minutes using a high-speed refrigerated benchtop centrifuge. The sample will separate into three layers: the lower organic phase, the middle layer, and the upper colorless aqueous phase (containing RNA). The volume of the aqueous phase is approximately 70% of the added Lysis Buffer volume.

5.Carefully transfer the supernatant (estimate the volume accurately) to a new user-prepared centrifuge tube. Add 1.5 volumes of absolute ethanol (must be at room temperature) and immediately pipette to mix. Precipitation may form after adding ethanol, but this will not affect the experiment. Do not centrifuge.

6.Load the mixture into the Spin AC RNA Micro Adsorption Column (placed in a collection tube) in batches (≤700 μL per aliquot). Centrifuge at 12,000 rpm for 30-60 seconds. Discard the filtrate in the collection tube and place the adsorption column back into the collection tube.

7.Add 700 μL of Wash Solution 1 (check if absolute ethanol has been added!). Centrifuge at 12,000 rpm for 30 seconds. Discard the filtrate and place the adsorption column back into the collection tube.

8.Add 500 μL of Wash Solution 2 (check if absolute ethanol has been added!). Centrifuge at 12,000 rpm for 30 seconds. Discard the filtrate and place the adsorption column back into the collection tube.

9.Repeat Step 8: Add another 500 μL of Wash Solution 2 (check if absolute ethanol has been added!). Centrifuge at 12,000 rpm for 30 seconds. Discard the filtrate and place the adsorption column back into the collection tube.

10.Place the RNA adsorption column back into the empty collection tube. Centrifuge at 13,000 rpm for 2-5 minutes to completely remove residual washing solution (residual ethanol in the washing solution may inhibit downstream reactions).
Note: This step is critical to remove residual ethanol from the adsorption column RS. Ethanol residue can affect subsequent enzymatic reactions (e.g., restriction digestion, PCR).

11.Transfer the RNA adsorption column to a new user-prepared RNase-free centrifuge tube. Add 20-30 μL of RNase-free H2O (heating in a 70-90℃ water bath can improve yield) to the center of the adsorption membrane according to the expected RNA yield. Incubate at room temperature for 1-2 minutes, then centrifuge at 13,000 rpm for 1 minute.
Remarks::For higher RNA concentration: Reapply the first eluate to the adsorption column and repeat Step 11.For expected RNA yield >10 μg: Add another 10-30 μL of RNase-free water and repeat Step 11, then combine the two eluates.Note: Eluting twice yields higher concentration RNA. Combining eluates from two separate elutions increases RNA yield by 15-30% compared to a single elution but results in lower concentration. Choose the method based on experimental needs.Higher elution volume leads to higher elution efficiency. To obtain higher RNA concentration, the elution volume can be appropriately reduced, but the minimum volume should not be less than 30 μL (excessively small volume reduces RNA elution efficiency and yield).