TRIzon Reagent

Item Number

T669996

• Storage Temp: Store at 2-8°C,Protected from light
• Shipped In: Wet ice

Product Description

Products

TRIzon is a broad-spectrum total RNA extraction reagent. It is fast and easy to use, with distinctive color and easy layering. It can be used to extract total RNA from animal tissues, plant material, various microorganisms and cultured cells, etc. Samples are fully lysed in TRIzon while maximizing RNA integrity. After centrifugation with chloroform, the solution splits into three layers: an upper colorless aqueous phase, an intermediate layer and a lower red organic phase, with the RNA distributed in the supernatant layer. After collecting the supernatant layer, the total RNA can be recovered by isopropanol precipitation, and the extracted total RNA has good integrity and is free of protein and DNA contamination, which 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, etc. The extracted total RNA can be used in a variety of molecular biology experiments, such as RT-PCR, RT-PCR, RT-PCR and in vitro translation.

Self-contained reagents: chloroform, isopropanol, 75% ethanol, RNase-free water (freshly opened or for RNA extraction).

Pre-experiment Preparation and Important Notes

1. To prevent RNase contamination, attention should be paid to the following aspects:

1) Use RNase-free plastics and tips to avoid cross-contamination.

(2) Glassware should be dry-roasted at 180°C for 4 hours before use, and plasticware can be soaked in 0.5M NaOH for 10 minutes, rinsed thoroughly with water and autoclaved.

3) RNase-free water should be used to prepare the solution.

(4) Operators wear disposable masks and gloves, and change gloves diligently during the experiment.

2. Avoid repeated freezing and thawing of the extracted samples, otherwise it will affect the rate and quality of RNA extraction.

3. This product contains phenol, which is toxic and corrosive. It can cause poisoning, burns, and other bodily injury if inhaled, in contact with skin, or swallowed. Protective items such as protective clothing, gloves, eye protection, face mask, etc. should be worn when using this product. In case of accidental contact with eyes, flush immediately with plenty of water and go to hospital for treatment.

4. After homogenization with TRIzon, samples can be left at -70°C for more than one month if chloroform is not added immediately.

5. RNA precipitation stored in 75% ethanol can be stored for one week at 2-8°C and for one year at -20°C. RNA has a relatively short half-life and is easily degraded, so it is recommended to carry out subsequent experiments as soon as possible after the extraction, e.g., reverse transcription into cDNA, Northern Blot etc.

If the downstream experiments are very sensitive to DNA, it is recommended that RNA be treated with RNase-free DNase I.

Procedure

1. Handling of various materials

1a. Plant tissues: take fresh plant tissues and grind them thoroughly in liquid nitrogen or cut them up and then put them directly in TRIzon

Grind quickly in, add 1 ml of TRIzon per 30-50 mg of tissue and mix well.

Note: The sample volume should generally not exceed 10% of the TRIzon volume.

1b. Animal tissues: Take fresh or -70℃ frozen animal tissues and cut them as much as possible, add 1ml TRIzon per 30-50 mg of tissue, homogenizer for homogenization. Or add 1ml of TRIzon after grinding in liquid nitrogen and mixing.

Note: The sample volume should generally not exceed 10% of the TRIzon volume.

1c. Monolayer culture cells: Aspirate off the culture solution, you can add appropriate amount of TRIzon directly into the culture plate (1ml TRIzon is needed for every 10 cm2 area), and blow repeatedly with a sampler to make the cells lysed. The cells can also be treated with trypsin, and then the cell solution can be transferred to RNase-Free centrifuge tubes, centrifuged at 300×g for 5 min, the cell precipitate was collected, and all the supernatant was carefully aspirated off, and 1 ml of TRIzon was added and mixed.

Note: 1) Do not collect more than 1 x 107 cells.

(2) The amount of TRNzol addition is determined by the culture plate area, not by the number of cells. Insufficient TRIzon dosage may result in DNA contamination in the extracted RNA.

3) Be sure to remove the cell culture fluid when collecting the cells, otherwise it will lead to incomplete lysis, resulting in a lower yield of RNA.

1d. cell suspension: collect cells by centrifugation. Add 1ml TRIzon per 5×106-1×107 animal, plant and yeast cells or per 107 bacterial cells.

Note: 1) Do not wash the cells before adding TRIzon to avoid RNA degradation.

2) Some yeast and bacterial cells may require homogenizer or liquid nitrogen grinding treatment.

1e.Blood treatment: Take fresh blood directly, add 3 times the volume of TRIzon (0.25 ml whole blood to 0.75 ml TRIzon is recommended) and mix with sufficient shaking.

1f. Optional step: For samples with a high content of proteins, fats, polysaccharides, or extracellular material, such as muscle tissue, adipose tissue, or plant tubers, the homogenization can be followed by centrifugation at 4°C, 12,000 rpm (~13,400 x g) for 10 minutes to remove insoluble material, where the precipitate contains extracellular material, polysaccharides, and high molecular weight DNA, with the RNA present in the supernatant.

2. Blow the sample several times repeatedly after adding TRIzon to the sample to fully cleave the sample. Leave at room temperature for 5 minutes to allow complete separation of the protein-nucleic acid complex.

3. Add chloroform to the above solution, 0.2 ml of chloroform for every 1 ml of TRIzon used, cap the tube, shake vigorously for 15 seconds and leave at room temperature for 2-3 minutes.

4.4°C 12,000 rpm centrifugation for 15 minutes, at this time the sample is divided into three layers: red organic phase, middle layer and upper colorless aqueous phase, the RNA is mainly in the aqueous phase, transfer the aqueous phase (about 600 μl) to a new RNase-Free centrifuge tube (self-provided).

5. Add an equal volume of isopropanol to the resulting aqueous phase solution, mix upside down and leave for 10 minutes at room temperature.

6.4°C 12,000 rpm centrifuge for 10 minutes, discard supernatant.

7. Wash the precipitate by adding 75% ethanol (prepared with RNase-free water). Wash the precipitate with 1 ml of 75% ethanol for every 1 ml of TRIzon used.

8. Centrifuge at 12,000 rpm for 3 minutes at 4°C and carefully aspirate the supernatant, taking care not to aspirate the RNA precipitate.

Note: The small amount of liquid remaining can be centrifuged briefly and then aspirated with a lance tip, taking care not to discard the precipitate.

9. Leave at room temperature for 2-3 minutes and dry. Add 30-100μl of RNase-free water to fully dissolve the RNA, and store the obtained RNA at -70°C to prevent degradation.

Note: Do not over dry the precipitate as it may be difficult to dissolve.