TRIzol NC Reagent

Self-provided reagents

Chloroform, isopropanol, 75% ethanol (prepared with RNase-free ddH₂O), RNase Free H₂O.

Ⅰ Preparation before experiment

The key to RNA preparation is to inhibit RNA degrading enzymes in cells and prevent contamination of RNA degrading enzymes in the equipment and reagents used. Therefore, the following measures must be taken in the experiment: wear disposable clean gloves and mask; use a special experimental bench for RNA operation; avoid talking during the operation, etc. The above methods can prevent the contamination of the experimenter's sweat and saliva by RNA degrading enzymes.

Cautions:

1. Try to use disposable enzyme-free sterile plastic consumables. If glassware is used, it should be treated with 0.1% DEPC aqueous solution at 37°C for 12 hours before use, and then autoclaved at 120°C for 30 minutes to inactivate residual DEPC.

2. Reagents used for RNA experiments must be sterilized by dry heat (180°C, 60min) or in glass containers after DEPC water treatment and sterilization using the above methods (disposable plastic containers for RNA experiments can also be used) , The sterile water used must be treated with 0.1% DEPC and then autoclaved.

3. Reagents and sterile water for RNA experiments should be dedicated to avoid cross-contamination after mixing.

4. This product is only for scientific research use, should not be used for clinical medical diagnosis and other irrational purposes.

5. This product contains phenol, which is toxic and corrosive. Protective items should be worn when using, such as protective clothing, gloves, eye masks, face masks, etc. In case of contact with eyes, rinse immediately with plenty of water and go to the hospital for treatment. In case of contact with skin, please rinse immediately with a large amount of detergent and water, if you still feel unwell, please go to the hospital for treatment.

Ⅱ Experiment Process

1. Sample Processing

1.1 Animal/plant tissues

1.1.1 Flash freeze fresh tissue in liquid nitrogen, and then quickly transfer to a mortar precooled with liquid nitrogen. Grind quickly with a pestle while adding liquid nitrogen until the sample is ground into powder thoroughly.

   Note: Insufficient grinding may impair RNA yield and quality.

1.1.2 Transfer the powdered sample into a centrifuge tube, add 500μL of TRIzol NC Reagent per 50mg of tissue, and vortex until the sample is completely lysed. Incubate at room temperature for 5min.

   Note: Up to 50mg of animal/plant tissue can be lysed per 500μL of TRIzol NC Reagent. Too much samples may lead to insufficient lysis and decrease product purity. Liver, spleen, kidney and other tissues are rich in DNA/RNA, and excessive sample inputs will lead to residual gDNA or low yield of RNA.

Note: If cryogenic grinding in liquid nitrogen is not feasible, mince the fresh tissue as finely as possible, immerse in the TRIzol NC Reagent, and homogenize in a high-speed electric homogenizer until the tissue pieces are completely lysed.

1.1.3 (optimize) Centrifuge at 11,200rpm (12,000xg) at room temperature for 5min. Carefully transfer supernatant into new 1.5ml centrifuge tube, do not absorb precipitate.

  Note: If the sample contains large amounts of protein, fat, polysaccharide or muscle fiber, plant tuber, etc., the insoluble substances can be removed by centrifugation. The precipitation obtained by centrifugation contains cell membrane, polysaccharide, high molecular weight DNA, while the RNA remains in the supernatant. When extracting tissue samples with a high fat content, if the upper layer contains a large amount of oil, it should be removed. Proceed to the next step after transferring the supernatant.

1.2 Suspension cells

1.2.1 Collect the cells by centrifugation, discard the supernatant thoroughly, and add 500μL of TRIzol NC Reagent per 1×10⁶ - 1×10⁷ cells.

1.2.2 Vortex or pipette up and down until the cells are completely lysed, and incubate at room temperature for 5min.

Note: For frozen cells, the addition of TRIzol NC Reagent should be immediately vortexed to avoid incomplete lysis.

1.3 Adherent cells

1.3.1 Discard the culture medium and wash once with 1×PBS.

1.3.2 Add 500μL TRIzol NC Reagent to each well of a 6-well cell culture plate or a 3.5cm diameter dish (approximately 10cm² culture area), allow the TRIzol NC Reagent to fully cover the cell layer, and then detach the cells by pipetting.

Note: Firmly adherent cells (cell clumps) can be detached with a cell scraper or a clean pipette tip, or add the volume of TRIzol NC Reagent up to 1mL. Alternatively, detach the cells using trypsin before adding TRIzol NC Reagent, and then follow the steps for suspension cells.

1.3.3 Transfer the mixture to a 1.5mL centrifuge tube, vortex or pipette up and down until the cells are completely lysed, and then incubate at room temperature for 5min.

2. RNA Extraction

2.1 Add Dilution Buffer to the above lysates.

2.1.1 Animal/plant tissues: Add 100μL Dilution Buffer per 500μL of TRIzol NC Reagent. Vortex thoroughly until the solution becomes a homogeneous emulsion. Incubate at room temperature for 5min.

2.1.2 Cells: Add 150μL Dilution Buffer per 500μL of TRIzol NC Reagent. Cap the centrifuge tube tightly and vortex thoroughly until the solution becomes a homogeneous emulsion. Incubate at room temperature for 5min.

   Note: Ensure that the solution should be fully mixed into a uniform solution, otherwise will impair the efficiency of RNA extraction and impurity removal.

2.2 Centrifuge at 11,200rpm (12,000×g) for 15min at room temperature.

2.3 Take out the centrifuge tube Carefully. At this time, impurities such as protein, DNA and polysaccharide in the sample precipitate to the bottom of the tube, RNA is distributed in the supernatant, and transfer the supernatant solution (about 500μL) carefully into a new centrifuge tube.

  Note: The upper aqueous phase, which takes up about 90% of the total volume, is approximately 500μL if 500μL of TRIzol NC Reagent is used for the extraction. Absorbing the underlying pellet will lead to genomic and impurity contamination.

Note: The supernatant may be slightly turbid or colored for some samples, which will not affect the product yield or purity. It is safe to proceed to the following steps.

Note: When the amount of tissue input is 50mg, the volume of the supernatant solution is recommended to be reduced to 450μl and avoid transferring any of the pellet.

2.4 Add an equal volume of isopropanol to the obtained supernatant. Mix well by inversion and incubate at room temperature for 10min.

2.5 Centrifuge at 11,200rpm (12,000×g) for 10min at room temperature. Then, gel-like RNA pellet can be seen at the side and bottom of the tube. Carefully discard the supernatant, be care not to lose the pellet.

  Note: Low content of RNA will lead to pellet invisible, so be careful to discard the supernatant to avoid losing the pellet.

  Note: To reduce the residue of impurities, discard the supernatant as far as possible in this step and do not lose the pellet.

2.6 Add 1mL of 75% ethanol (prepared with RNase-free ddH₂O). Gently flick the tube to resuspend the pellet, and invert the tube a few times.

2.7 Centrifuge at 11,200rpm (12,000xg) at room temperature for 3min. Discard the supernatant, be care not to lose the pellet.

2.8 Repeat Step 6 and 7 once. Discard the supernatant.

Note: In order to reduce the residual supernatant, the supernatant should be discarded as much as possible. It is recommended to discard most of the supernatant, briefly centrifuge all the liquid to the bottom of the tube, and then use a 10μL pipette to suck off the remaining liquid, taking care not to loss pellet.

2.9 Air dry at room temperature in clean bench. Add 20-100μL of RNase-free ddH₂O to dissolve the pellet and vortex at room temperature for 3 min (or pipette up and down) for dissolve thoroughly. The RNA can be aliquoted and stored at -85 ~ -65°C for long-term storage or -30 ~ -15°C for short-term storage.

  Note: Air dry the RNA pellet for 2 - 3 min. Do not over dry the pellet because completely dry RNA will be difficult to resuspend. The RNA should be completely resuspended, or the concentration quantitation may be inaccurate.

Ⅲ FAQ & Troubleshooting