Products

Product Introduction：
TRI LS Reagent is a reagent for directly isolating total RNA from liquid samples derived from humans, animals, plants, yeast, bacteria, and viruses. It can also extract total RNA from samples such as animal tissues, plant materials, various microorganisms, and cultured cells. This method achieves excellent separation results for small amounts of tissue (50-100 mg), cells (5×10⁶), as well as large amounts of tissue (≥1 g) and cells (>10⁷). While samples are fully lysed in TRI LS Reagent, the integrity of RNA is maximally preserved. After adding chloroform and centrifuging, the solution separates into three layers: an upper colorless aqueous phase, an interphase, and a lower organic phase. RNA is distributed in the upper aqueous phase. Total RNA can be recovered by precipitating the collected aqueous phase with isopropanol. The extracted total RNA has good integrity, no protein or DNA contamination, and can be used in various routine molecular biology experiments, such as RT-PCR, Real-time RT-PCR, Northern blot, Dot Blot, and in vitro translation.
TRI LS Reagent promotes the precipitation of various RNAs of different molecular weights from different species. For example, RNA extracted from rat liver, subjected to agarose gel electrophoresis and stained with ethidium bromide, shows multiple discrete high-molecular-weight bands (mRNA and hnRNA components) between 7 kb and 15 kb, two dominant ribosomal bands (~5 kb for 28S) and ~2 kb for 18S, and low-molecular-weight RNA between 0.1 and 0.3 kb (tRNA, 5S). When the extracted RNA is diluted with TE, its A260/A280 ratio is ≥1.8. Note that in conventional agarose gel electrophoresis, the 28S band is approximately at 2 kb and the 18S band at around 1 kb, with significant variations in positions depending on gel concentration.

Application Scope：
Suitable for extracting total RNA from liquid samples of humans, animals, plants, yeast, bacteria, and viruses.

Operation Steps：
Note: Wear gloves and eye protection when extracting RNA with TRI LS Reagent. Avoid contact with skin and clothing. Perform operations in a chemical fume hood to prevent inhalation. Unless otherwise specified, all operations should be carried out at room temperature (15~30°C).

1.Homogenization
a.Biological Fluids:
Add 0.75 mL TRI LS Reagent to 0.25 mL liquid sample (serum, plasma, cerebrospinal fluid, etc.). Pipette the liquid sample several times to assist in lysing cells within the sample. Add at least 0.75 mL TRI LS Reagent per 5~10×106 cells. The final volume ratio of TRI LS Reagent to liquid sample must always be 3:1.

b.Animal Tissues:
Homogenize tissue samples with a glass or high-power homogenizer. Add 0.75 mL TRI LS Reagent to 50~100 mg tissue or 0.25 mL tissue suspension. Generally, the volume of 50~100 mg tissue is less than 0.25 mL. If the tissue sample volume is less than 0.25 mL, adjust the volume to 0.25 mL with sterile water to maintain the 3:1 volume ratio.

c.Adherent Cells (Monolayer Culture):
Directly add 0.3 mL-0.4 mL TRI LS Reagent to a 3.5 cm diameter culture dish to lyse cells. Pipette to ensure complete lysis. The amount of TRI LS Reagent required is determined by the culture dish area rather than the number of cells (add 0.3-0.4 mL per 10 cm²). No additional water is needed for the lysate, as residual culture medium adhering to the dish sufficiently dilutes the TRI LS Reagent.
Note: Adherent cells may not completely detach from the culture flask/dish, which does not indicate incomplete lysis. At this point, the cell membrane has actually ruptured completely, and RNA has been released. Proceed with the subsequent steps.

d.Cell Suspensions:
Pellet cells by centrifugation. Lyse cells by repeated pipetting in TRI LS Reagent. Add 0.75 mL TRI LS Reagent per 5~10×106 animal cells, plant or yeast cells, or per 1×107 bacteria. Adjust the sample volume to 0.25 mL with sterile water as described in Step b. Avoid washing cells before adding TRI LS Reagent, as this increases the risk of mRNA degradation. Homogenization may be required to lyse certain yeast and bacterial strains.

e.Plant Tissues:
Grind fresh plant tissue thoroughly in liquid nitrogen, or chop the plant tissue and grind rapidly directly in TRI LS Reagent. Add 0.75 mL TRI LS Reagent to 50-100 mg tissue. Adjust the sample volume to 0.25 mL with sterile water as described in Step b, and mix well.

2.Vigorously vortex the homogenized sample and incubate at room temperature for 5 minutes to fully dissociate ribosomes.

3.Optional Step:Centrifuge at 12,000 rpm for 10 minutes at 4°C, then collect the supernatant.
This step is recommended for samples with high protein, fat, polysaccharide content, or for tissues such as muscle and plant tubers/nodules to remove impurities. The pellet after centrifugation contains cell membranes, polysaccharides, and high-molecular-weight DNA, while the supernatant contains RNA. For adipose tissue samples, remove the upper layer of excess oil. Proceed with the clear homogenate for the next step.

4.Add 0.2 mL chloroform to 0.75 mL TRI LS Reagent. Tighten the tube cap, vortex vigorously for 15 seconds, and incubate at room temperature for 2~3 minutes.

5.Centrifuge at 12,000 rpm for 10-15 minutes at 4°C in a high-speed refrigerated centrifuge. After centrifugation, the mixture separates into three layers: the lower organic phenol-chloroform phase, the interphase, and the upper colorless aqueous phase. RNA is exclusively present in the aqueous phase. The volume of the aqueous phase is approximately 70% of the added TRI LS Reagent volume. (The organic phase and interphase contain proteins and DNA; contact us for extraction methods if needed.)

6.Transfer the aqueous phase to a clean centrifuge tube, add an equal volume of isopropanol. Invert to mix and incubate at room temperature for 10 minutes. RNA pellets are usually invisible before centrifugation but form gelatinous precipitates on the tube side and bottom after centrifugation.

7.Centrifuge at 12,000 rpm for 10 minutes at room temperature or 4°C, then discard the supernatant.

8.Wash the pellet with 75% ethanol. Use 1 mL 75% ethanol per 0.75 mL TRI LS Reagent.

9.Centrifuge at 12,000 rpm for 3 minutes at room temperature or 4°C, then discard the supernatant. Be careful not to lose the RNA pellet.
Note: For any remaining residual liquid, centrifuge briefly and aspirate with a pipette, taking care not to remove the pellet.

Air-dry at room temperature for 2-3 minutes. Add 30-100 μL RNase-free water to fully dissolve the RNA. Store the extracted RNA at -70°C to prevent degradation. Note: Do not over-dry the pellet, as this will make it difficult to dissolve.