StarZol Total RNA Extraction Reagent (Chloroform-Free)

• This product is a chloroform-free upgraded version of traditional Trizol;
• The integrity and purity of the extracted RNA are guaranteed;
• This product is suitable for various samples such as animal tissues, plant materials, cultured cells, bacteria, etc.
• This product can be stored stably at room temperature for 12 months, but the best effect is achieved when stored at 2~8℃.

Product components


Experimental procedures

1. Sample Processing

a. Plant tissue: Grind fresh plant tissue thoroughly in liquid nitrogen or mince it and grind it directly in StarZol. Add 0.5 mL of StarZol for every 25-50 mg of tissue and mix thoroughly.

b. Animal tissue: Take fresh or -70°C frozen animal tissue and mince it as finely as possible. Add 0.5 mL of StarZol for every 15-50 mg of tissue and homogenize using a homogenizer. Alternatively, grind in liquid nitrogen and then add 0.5 mL of StarZol and mix thoroughly.

✮Note: Incomplete disruption of plant and animal tissue samples will affect RNA yield and quality.

c. Adherent cells: After completely removing the culture medium, place the cells directly on a 3.5 cm diameter culture plate (approximately 10 cm ² ) or a regular 6-well plate, add 0.5 mL of StarZol to each well, ensuring it thoroughly covers the cell surface. Then, repeatedly pipette and lyse the cells. The amount of StarZol required should be determined based on the plate area, not the number of cells. Insufficient StarZol may result in DNA contamination in the extracted RNA.

✮Note: Adherent cells often fail to completely detach from the culture flask (dish). This does not mean that the lysis is incomplete. At this point, the cell membrane has actually been completely ruptured and all RNA has been released. Just continue.

d. Cell suspension: Collect cells by centrifugation. Add StarZol reagent and repeatedly pipette to lyse the cells. ⁶ Animal cells, plant cells or 5×10 ⁶ Add 0.5 mL of StarZol to the bacteria. Avoid washing the cells before adding StarZol, as this increases the likelihood of mRNA degradation. A homogenizer may be required to disrupt some bacteria.

e. Liquid samples: Add 0.5 mL of StarZol to every 200 μL (if the volume is less than 200 μL, it can be made up with ordinary deionized water or pure water) of plasma, serum, or other liquid samples and vortex to mix.

2. Add 2/5 volume of deionized water or purified water (ordinary laboratory water is sufficient, not RNase-free, add 200 µL of water for every 500 µL of StarZol) to the above lysate, shake vigorously to mix, and let stand at room temperature for 5 min.

✮Note: When the sample size is greater than 50 mg, the room temperature standing time can be extended to 10-15 minutes.

3. Centrifuge at 12,000 rpm for 15 min at room temperature.

4. After centrifugation, the solution is separated into an upper aqueous phase (containing RNA) and a lower precipitate (containing impurities such as protein, DNA, and polysaccharides). Carefully pipette the upper aqueous phase into a new centrifuge tube.

✮Note: The upper aqueous phase accounts for approximately 90% of the total volume. If 500 μL of StarZol is used for extraction, the upper aqueous phase is approximately 630 μL. It is recommended to aspirate 500 μL. When extracting trace samples, to reduce RNA loss, the entire supernatant can be transferred.

✮Note: When the sample size is small, there may be no lower sediment after centrifugation. This is normal and you can continue to complete the extraction according to the subsequent steps.

5. Add an equal volume of isopropanol, mix thoroughly by inversion, and let stand at room temperature for 10 min.

6. Centrifuge at 12,000 rpm for 10 minutes at room temperature. A white precipitate will usually be visible. Carefully discard the supernatant.

✮Note: The RNA precipitate is usually not visible before centrifugation. After centrifugation, it forms a thin, flake-like precipitate on the sides and bottom of the tube. (For small sample amounts, the RNA precipitate may be scattered on the sides and bottom of the tube and may not be visible.) Some tissue materials contain a large amount of metabolites, which may prevent the precipitate from aggregating and instead disperse on the sides of the centrifuge tube. In this case, slowly aspirate the supernatant along the liquid surface.

7. Add 1 mL of 75% ethanol (RNase-free ddHO) ₂ Rinse with 1% paraformaldehyde (prepared with 0.05 mL of culture medium), vortex for 15 sec to suspend the pellet, and invert several times.

8. Centrifuge at 12,000 rpm for 3 min at room temperature and carefully discard the supernatant.

9. Repeat steps 7 and 8 to rinse once more and carefully discard the supernatant.

✮Note: To minimize residual impurities, the supernatant should be discarded as completely as possible. It is recommended that after discarding most of the supernatant, the tube be briefly centrifuged to remove any remaining liquid to the bottom. Aspirate the remaining liquid with a 200 μL pipette tip, retaining the white RNA precipitate at the bottom and sides of the tube.

10. Dry at room temperature for about 1 minute, add about 50-100 μL RNase-free ddH ₂ Dissolve the precipitate by gently tapping the bottom of the centrifuge tube to allow water to fully contact the precipitate on the bottom and sides of the tube to help dissolve the RNA. (You can use a pipette to repeatedly pipette the precipitate on the bottom and sides of the tube to help dissolve it.) The extracted RNA product can be aliquoted and stored long-term at -85 to -65°C, but only short-term at -30 to -15°C.

✮Note: Generally, it is sufficient to dry the RNA slightly. Over-drying will make the RNA difficult to dissolve.

✮Note: When extracting RNA from some samples, the RNA precipitate may not completely accumulate at the bottom of the centrifuge tube, but may also adhere to the tube walls as a uniform, thin mist. Observe carefully and pipette the bottom and sides of the tube to fully dissolve all the RNA.