Introduction
The EZ-press microRNA Purification Kit PLUS provides a simple, reliable, and rapid method for isolating high-quality miRNA ( total RNA including miRNA, mRNA, lncRNA, CircRNA ) from a wide variety of sources, without the need for toxic substances such as phenol or chloroform.
This Kit can be used with cultured cells and part of animal tissues. The purified miRNA is suitable for use in a variety of downstream applications, including: RT-PCR, RT-qPCR, Northern blotting, nuclease protection assays, RNA sequencing and so on.
Biological samples are first lysed and homogenized in a strong denaturant containing buffer, which immediately inactivates RNases to ensure isolation of intact miRNA.
After homogenization, the lysate is passed through the DNA Removing Spin Column which traps the DNA on the column, while miRNA can pass through the column with the flow-through.
Then ethanol is added to the miRNA containing flow-through. The mixture is then passed through the RNA binding Spin Column to which the miRNA binds. Impurities are effectively removed by subsequent DNase treatment and washing.
The purified miRNA is then eluted in Elution Buffer and may be used in a variety of downstream applications.
Experimental Procedure at a Glance
Protocol
Sample Homogenization
1A. For adherent cells ≤ 3 × 106/sample:
a) Remove the growth medium from the cells.
b) Wash cells with appropriate volume of PBS.
c) Add 500 μl of Lysis Buffer. Pipette up and down for 10 times to suspend the cells.
d) Transfer the cell lysate to a new tube and vortex for 10 seconds at high speed to completely lyse the cells.
1B. For adherent cells with a cell number of more than 3×106/sample or suspension cells:
(For adherent cells only, for suspension cells, start at step b)
a) Detach cells using the sub-culturing method routinely employed in your laboratory. Pellet 1 × 106 cells in a 1.5 ml centrifuge tube by centrifugation at 2000 × g for 1 minute.
b) Completely remove the supernatant by aspiration.
c) Add 500 μl of Lysis Buffer.
d) Vortex for 10 seconds at high speed to completely lyse the cells.
1C. For animal tissues:
For most tissue miRNA purification, EZB-miRN1 Kit is suggested.DNA Depleting
Transfer the homogenized lysate of step 1 to the DNA Removing Spin Column.
Centrifuge at 4000 × g for 1 minute. Transfer the miRNA containing flow-through to a new RNase free 1.5 ml centrifuge tube for the isolation of miRNA.
RNA Binding
Add 1.6 volume of ethanol to each volume of the flow-through (See step 3. There may be precipitates at this step. This is a normal phenomenon).
Pipette up and down for several times to disperse the precipitates, and transfer the sample to the RNA Spin Column. Centrifuge at 4000 × g for 1 minute.
DNase Treatment
Mix 2 μl gDNA Remover with 10 μl ddH2O, and add to the center of the column. Incubate at room temperature for 5 minutes to degrade the DNA.
RNA Column Washing
Add 500 μl of Wash Buffer to the RNA column. Centrifuge at 12000 × g for 1 minute.
Transfer the column to an RNase free 1.5 ml centrifuge tube, open the lid and keep in the air for 2 minutes.
RNA Elution
Add 20 ~ 30 μl of Elution Buffer to the center of the RNA column and incubate at room temperature for 1 minute.
Centrifuge at 12000 × g for 1 minute.
Discard the column，determine the RNA concentration, do the following experiment with the purified RNA, or store the RNA at -80°C until needed.
Representative experimental results
Detection of microRNA level in A549 cells by RT-qPCR (the microRNA was purified by EZ-press microRNA Purification Kit plus). The figure below shows that both the miR-145 and U6 can be amplified efficiently and specially.
For detailed information, please check the product manual.
