Products

Tissue/Cell 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. This kit uses a unique lysis buffer/β-mercaptoethanol to rapidly lyse cells 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.Rapid and simple operation: processing of a single sample can generally be completed within 30 minutes.
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 rapid extraction of miRNAs and other small RNAs from various cells and tissues.

Operation Steps:
Note: Before first use, add the specified volume of absolute ethanol to the 70% Ethanol bottle, Wash Solution 1 bottle, and Wash Solution 2/3 bottle!

1.Cultured Cells
a.Collect <10⁷ suspension cells into a 1.5 mL centrifuge tube. (For adherent cells cultured in plates or flasks: lyse directly. Aspirate all residual culture medium as much as possible, add 1 mL of Lysis/Binding Buffer, gently shake immediately to ensure full contact between the buffer and all cells at the bottom of the vessel to lyse cells and inactivate RNases. Gently pipette up and down to mix, then proceed to Step 3 below.)
b.Centrifuge at 13,000 rpm for 10 seconds (or 300 g for 5 minutes) to pellet the cells. Aspirate and discard the supernatant completely, leaving the cell pellet. Incomplete removal of the supernatant will dilute the lysis buffer, resulting in reduced yield and purity.
c.Flick the tube wall to fully resuspend the cell pellet. Add 1 mL of Lysis/Binding Buffer, vortex or pipette up and down to lyse thoroughly and mix well.
d.Proceed to Step 3 in the general operation steps below.

2.Animal Tissues (e.g., Mouse Liver, Brain)
a.Quickly cut fresh tissue into small pieces with a scalpel. Add 1 mL of Lysis/Binding Buffer per 50-100 mg of tissue, then homogenize thoroughly manually or with an electric homogenizer. Alternatively, grind the tissue into fine powder in liquid nitrogen, transfer an appropriate amount (approximately 50-100 mg) of tissue powder into a 1.5 mL centrifuge tube containing 1 mL of Lysis/Binding Buffer, and vortex vigorously to mix well.
b.Optional (generally not required): For large sample volumes, obvious particles or insolubles, excessive viscosity, or incomplete lysis, immediately pass the lysate through a disposable 5 mL syringe with a needle (approximately 0.9 mm) 10 times (or homogenize electrically for 30 seconds) to shear DNA, reduce viscosity, and improve yield.
c.Proceed to Step 3 in the general operation steps below.

General Operation Steps
3.Incubate at room temperature for 5 minutes to fully separate nucleic acid-protein complexes.
4.Add 200 μL of chloroform and vortex vigorously for 15 seconds.
5.Incubate at room temperature for 2-3 minutes, then centrifuge at 13,000 rpm for 10 minutes.
6.Carefully transfer the supernatant (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.
7.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.
8.Add 700 μL of Wash Solution 1 (check if absolute ethanol has been added!). Centrifuge at 12,000 rpm for 30 seconds and discard the filtrate.
9.Add 500 μL of Wash Solution 2/3 (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.
10.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).
11.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.
12.If the expected RNA yield is >30 μg:Option 1: Add another 30-50 μL of RNase-free water and repeat Step 11, then combine the two eluates.Option 2: Reapply the first eluate to the adsorption column and repeat Step 11 (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.