The EZBioscience® EZ-press RNA Purification Kit provides a simple, reliable, and rapid method for isolating high-quality total RNA from a wide variety of sources, without the need for toxic substances such as phenol or chloroform.
The EZ-press RNA Purification Kit can be used with cultured cells and animal tissues.
The purified total RNA is suitable for use in a variety of downstream applications, including: RT-PCR, RT-qPCR, Northern blotting, nuclease protection assays,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 RNA.
After homogenization, ethanol is added to the sample. The lysate is then passed through an RNA binding Spin Column containing a silica-based membrane to which the RNA binds. Impurities are effectively removed by subsequent washing.
The purified total RNA is then eluted in Elution Buffer and may be used in a variety of downstream applications.
Experimental Procedure at a Glance
Protocol
Sample Homogenization
Note: Before using for the first time, add 48 ml of 100% ethanol to the Wash Buffer.
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 make the cells completely lysed.
1B. For cultured suspension cells or adherent cells > 3×106/sample:
a) (For adherent cells only, for suspension cells, start at step b) Detach cells using the subculturing method routinely employed in your laboratory.
b) Pellet 1 × 106cells in a 1.5 ml centrifuge tube by centrifugation at 2,000 × g for 1 minute.
c) Completely remove the supernatant by aspiration.
d) Add 500 μl of Lysis Buffer.
e) Vortex for 10 seconds at high speed to to make the cells completely lysed.
1C. For animal tissues:
a) Place 1 ~ 30 mg tissue in a 1.5 ml centrifuge tube.
b) Add 500 μl of Lysis Buffer.
c) Homogenize the tissue with a pestle or rotor-stator homogenizer.
RNA Binding
2. Add equal volume of 100% ethanol to each volume of cell or tissue homogenate.
3. Mix thoroughly and transfer the sample to the Spin Column. Centrifuge at 4,000 × g for 1 minute.
Column Washing
4. Add 500 μl of Wash Buffer to the column. Centrifuge at 12,000 × g for 1 minute.
5. 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
6. Add 20 ~ 50 μl of Elution Buffer to the center of the column and incubate at room temperature for 1~2 minute.
7. Centrifuge at 12,000 × g for 1 minute (Optional: transfer the eluate back to the column, wait for 5min and repeat centrifuge once again will get more RNA).
8. Discard the column，test the RNA concentration, do the following experiment with the purified RNA, or store the RNA at -80 °C until needed.
Representative experimental results
Figure 1. RNA isolated from different amounts of A549 cells using EZBioscience® EZ-press RNA Purification Kit and TRIzol. M: 1kb DNA Ladder; Lanes 1 ~ 3: EZ-press RNA Purification Kit; Lanes 4 ~ 6: TRIzol; 1: 2 × 105 cells; 2: 4 × 105 cells; 3: 6 × 105 cells; 4: 2 × 105 cells; 5: 4 × 105 cells; 6: 6 × 105 cells. These results showed that more RNA can be isolated from the same amount of cells by EZBioscience® EZ-press RNA Purification Kit than TRIzol.
Figure 2. Detection of mRNA level of 4 genes (ACTIN, P53, SNAIL and ZO-1) in A549 cells by RT-qPCR (the RNA was purified by EZ-press RNA Purification Kit and TRIzol method respectively). Figure 2. Shows that, the Ct values of these genes in the RNA sample purified by EZ-press RNA Purification Kit are highly correlated with the RNA purified by TRIzol method (R2=0.9898). And the Ct values from the RNA derived by EZ-press RNA Purification Kit are smaller than by the TRIzol method.
These data from Figure 1. and Figure 2. indicate that, EZ-press RNA Purification Kit can be a good substitution for the TRIzol method in RNA purification. And also, using this Kit can get better results than the TRIzol method.
For detailed information, please check the product manual.
