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 stably stored at room temperature for 12 months, but the best effect is achieved when stored at 2~8℃.

Product composition


Experimental procedures

1. Sample processing

a. Plant tissue: Grind fresh plant tissue thoroughly in liquid nitrogen or cut it into small pieces and grind it quickly in StarZol. Add 0.5 mL StarZol for every 25 - 50 mg of tissue and mix well.

b. Animal tissue: Take fresh or -70℃ frozen animal tissue and chop it into small pieces as much as possible, add 0.5 mL StarZol for every 15-50 mg of tissue, and homogenize it with a homogenizer. Or grind it in liquid nitrogen and add 0.5 mL StarZol to mix well.

✮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 add 0.5 mL StarZol to each well of a regular 6-well plate to fully cover the cell surface, then repeatedly pipette to lyse the cells. The amount of StarZol required is determined based on the area of the culture plate rather than the number of cells. Insufficient StarZol may result in DNA contamination in the extracted RNA.

✮Note: Adherent cultured cells often cannot completely fall off from the culture flask (dish), which does not mean that the lysis is incomplete. At this time, the cell membrane has actually been completely ruptured and all RNA has been released, so you can 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 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: For every 200 μL (if less than 200 μL, ordinary deionized water or pure water can be used to make up) of plasma, serum and other liquid samples, add 0.5 mL StarZol and oscillate to mix.

2. Add 2/5 volume of deionized water or pure water (ordinary laboratory water is fine, RNase-free is not required, add 200 µL water for every 500 µL 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 min.

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

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

✮Note: The upper aqueous phase accounts for about 90% of the total volume. If 500 μL StarZol is used for extraction, the upper aqueous phase is about 630 μL. It is recommended to draw 500 μL. When extracting trace samples, in order to reduce RNA loss, all supernatants can be transferred.

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

5. Add an equal volume of isopropanol, invert to mix, and let stand at room temperature for 10 min.

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

✮Note: RNA precipitates are usually not visible before centrifugation. After centrifugation, thin flakes of precipitates are formed on the side and bottom of the tube (when the sample amount is small, the RNA precipitates are scattered on the side and bottom of the tube and it is possible that no obvious precipitates can be seen). Some tissue materials contain more metabolites, resulting in the precipitates not being able to aggregate but being scattered on the wall of the centrifuge tube. At this time, please slowly aspirate the supernatant along the liquid surface.

7. Add 1 mL 75% ethanol (RNase-free ddHO) ₂ O preparation), rinse, vortex for 15 sec to suspend the precipitate, 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 reduce residual impurities, the supernatant should be discarded as cleanly as possible. It is recommended to discard most of the supernatant, centrifuge briefly to shake the remaining liquid to the bottom of the tube, and use a 200 μL pipette to suck out the remaining liquid, leaving the white RNA precipitate at the bottom and side of the tube.

10. Dry at room temperature for about 1 min, add about 50-100 μL RNase-free ddH ₂ O to dissolve the precipitate, flick the bottom of the centrifuge tube to allow water to fully contact the precipitate on the bottom and side of the tube to help dissolve the RNA (you can use a pipette to repeatedly blow the precipitate on the bottom and side of the tube to help dissolve it), so that the RNA precipitate is fully dissolved. The extracted RNA product can be packaged and stored at -85 ~ -65℃ for a long time, and can only be stored at -30 ~ -15℃ for a short time.

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

✮Note: When extracting RNA from some samples, the RNA precipitate is not completely gathered at the bottom of the centrifuge tube, but is also adsorbed on the side wall of the tube in a uniform mist-like precipitate. Please observe carefully and use a pipette to blow the bottom of the tube and the side wall of the tube where the precipitate is located to fully dissolve all the RNA.