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Blood/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:
Recent extensive research on RNA interference and regulatory small RNAs has created an urgent need for a kit capable of efficiently extracting RNAs of approximately 15-30 nucleotides in size, including siRNAs and miRNAs. However, traditional RNA extraction methods such as silica membrane adsorption fail to effectively recover small RNAs, and phenol/guanidine extraction combined with ethanol precipitation cannot efficiently precipitate and recover small molecular RNAs. Extraction from blood samples is particularly challenging due to their unique characteristics.
This kit uses a unique lysis buffer to directly and rapidly lyse whole blood (liquid samples) and inactivate cellular RNases. Intense organic extraction removes proteins and DNA, allowing RNAs (including small molecular RNAs) to bind to the special silica matrix membrane in the spin column. Through a series of rapid washing-centrifugation steps, washing solutions further remove impurities such as cellular metabolites and proteins. 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.Eliminates ethanol precipitation steps that easily result in loss of small molecular RNAs.
3.Unique lysis buffer formulation enables direct lysis of whole blood without prior red blood cell removal.
4.Multiple column washing steps ensure high purity, with a typical OD260/OD280 ratio of 1.9-2.0 and almost no DNA residue. Suitable for RNAi, RT-PCR, Northern blot, and various other experiments.

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

Operation Steps:
Note: Before first use, add the specified volume of absolute ethanol to the 70% Ethanol bottle, Wash Solution 1 (BR1) bottle, and Wash Solution 2/3 (BR2) bottle, mix thoroughly. Mark the corresponding box immediately after adding ethanol to avoid repeated additions!

1.Add 0.75 mL of Lysis Buffer to 0.25 mL of liquid sample (serum, plasma, cerebrospinal fluid, etc.). Pipette the liquid sample several times to assist in lysing cells in the sample. Add at least 0.75 mL of Lysis Buffer per 5-10×10⁶ cells. For samples with high contaminants (e.g., whole blood samples), dilute 1:1 with sterile water before extraction. The final volume ratio of Lysis Buffer to liquid sample must be 3:1.

2.Vortex the sample vigorously to mix, then incubate at 15-30°C for 5 minutes to completely dissociate nucleoprotein complexes.

3.Add 0.2 mL of chloroform per 0.75 mL of Lysis Buffer, vortex vigorously for 15 seconds, and incubate at room temperature for 2 minutes.

4.Centrifuge at 12,000 rpm at 4℃ for 10 minutes. 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, approximately 600 μL) to a new centrifuge tube. Add 1.5 volumes of absolute ethanol (must be at room temperature, usually 900 μL) and vortex to mix. Precipitation may occur, but this does not affect the extraction process. Mix immediately by pipetting; do not centrifuge, and proceed to the next step immediately.

6.Load the mixture (≤700 μL per aliquot; load in two batches if necessary) into a Spin RA Adsorption Column (placed in a collection tube). Centrifuge at 12,000 rpm for 30-60 seconds and discard the filtrate.

7.Add 700 μL of Wash Solution 1 (BR1) (check if absolute ethanol has been added!). Centrifuge at 12,000 rpm for 30 seconds and discard the filtrate.

8.Add 500 μL of Wash Solution 2/3 (BR2) (check if absolute ethanol has been added!). Centrifuge at 12,000 rpm for 30 seconds and discard the filtrate. Repeat this step with another 500 μL of Wash Solution 2/3 (BR2).

9.Place the Spin RA Adsorption Column back into the empty collection tube. Centrifuge at 13,000 rpm for 2 minutes to completely remove residual washing solution (residual ethanol in the washing solution may inhibit downstream reactions).

10.Transfer the Spin RA Adsorption Column to a new RNase-free centrifuge tube. Add 30-50 μL of RNase-free water (preheating at 100°C in a water bath for better results) to the center of the adsorption membrane according to the expected RNA yield. Incubate at room temperature for 1 minute, then centrifuge at 12,000 rpm for 1 minute.

11.If the expected RNA yield is >30 μg:Option 1: Add another 30-50 μL of RNase-free water and repeat Step 10, then combine the two eluates.Option 2: Reapply the first eluate to the adsorption column and repeat Step 10 (for higher RNA concentration).
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.