Trizol is a reagent for extracting total RNA from cells or tissues. This product adopts the same principles and methods as TRlzol from Invitrogen, with identical extraction methods and steps. The color of Trizol is the same as that of TRIzol. After adding chloroform, the upper layer appears colorless and the lower layer appears red, making it easy to absorb the upper aqueous phase. The RNA extracted by Trizol is free from DNA and protein contamination. The A260/280 value of RNA obtained by dissolving it in DEPC water is generally 1.8-2.0. When cells are lysed or homogenized with tissues, Trizol can maintain the integrity of RNA in the sample, effectively inhibiting RNA degradation. 5-15 μ g RNA can be obtained by extracting 1x106 cells with Trizol; Extraction with Trizol can yield 1-10 μ g RNA per mg of tissue. The yield varies depending on the cells and tissues. The RNA extracted by Trizol can be directly used for Northern blotting, point hybridization, mRNA purification, in vitro translation, RNase protection assay, cDNA cloning, and RT-PCR; It can also be used for gene expression chip analysis, high-throughput sequencing, and other situations that require high RNA quality.
Precautions：
1. If it is necessary to measure the A260/A280 ratio of RNA, it is recommended to dilute the sample with RNA quantification buffer for measurement.
2. All centrifuge tubes, gun heads, and related solutions must be free of RNAse contamination. High temperature resistant objects can be baked at 180°C for 4 hours to remove RNA enzymes. For other objects, RNA enzymes can be removed by 1. soaking them overnight in 0.01% DEPC water, followed by sterilization and drying. The solution needs to be prepared with DEPC water. Add 0.01% DEPC to re distilled water or MiliQ grade water, treat overnight, and sterilize to obtain DEPC water.
3. The effectiveness of extracting total RNA from frozen cells or tissues is usually inferior to that of fresh cells or tissues. Because during the freezing and thawing process of cells or tissues, some RNases within the cells or tissues are released and the samples are sheared. If RNA cannot be extracted in a timely manner, it is recommended to add an appropriate amount of Trizol first, lyse the sample, and then freeze it.
4. Disposable gloves must be worn during operation, and try not to exhale or speak towards RNA samples to prevent RNA enzyme contamination. It is recommended to wear a disposable mask during operation.
5. Trizol contains toxic substance phenol, avoid contact with skin or inhalation. To prevent splashing into the eyes, please wear protective glasses or use a transparent protective screen. If skin comes into contact with Trizol, immediately rinse with a large amount of detergent and water. If discomfort persists, seek medical advice.
Instructions for Use：
1 Sample slurry
1.1 Determine the type of sample and homogenize it at room temperature according to the table below. The volume of the sample cannot exceed 10% of Trizol. It is necessary to ensure the use of sufficient amounts of Trizol, as insufficient amounts of Trizol can lead to DNA contamination of RNA.
Type
| process
|
organization
| 1. 50-100mg tissue+1mL Trizol;
2. Use a homogenizer to homogenize the sample.
Attention: After collecting the samples, conduct the experiment immediately or freeze them immediately.
|
Adherent cell
| 1. Remove the culture medium;
2. Add 1mL Trizol to every 10cm2 culture dish;
Note: Add 1mL Trziol to a 35mm culture dish, 3mL Trziol to a 60mm culture dish, and 8mL Trziol to a 100mm culture dish, regardless of the number of cells;
3. Use a pipette tip to repeatedly blow and lyse cells in a culture dish.
|
Suspension cell
| 1. After collecting cells by centrifugation, remove the culture medium;
2. Add 0.75mL Trizol to 0.25mL sample (5-10x106cells, animal, plant or yeast, or 10x107 bacterial cells);
Note: Do not wash cells before adding Trizol to avoid increasing the possibility of mRNA degradation;
3. Repeatedly blow and beat to lyse cells, yeast and bacterial cells need to be homogenized. |
1.2 (Optional) When preparing RNA from samples containing a large amount of fat, protein, polysaccharides, or extracellular matrix (such as muscle, fat, or plant tissue), an additional centrifugation step is required to remove insoluble substances from the sample. Attention: If DNA separation is performed, do not centrifuge.
Type
| process
|
Tissues or cells high in fat, protein, polysaccharides, or extracellular matrix
| 1. After homogenization, centrifuge at 12000g for 10 minutes at 4 ° C;
Note: The precipitate contains extracellular matrix, polysaccharides, and high molecular weight DNA, while the supernatant contains RNA. In high-fat samples, the upper layer of the supernatant is the fat layer;
2. Remove the fat layer;
3. Transfer the supernatant to a new test tube. |
2 Phase separation:
2.1 Incubate the homogenate at room temperature for 5 minutes to allow separation of the nuclear protein complex;
2.2 Add 0.1mL of phase separation reagent BCP or chloroform to every 1mL of Trizol prepared slurry, and cover tightly;
2.3 Vigorously shake the test tube for 15 seconds;
2.4 Incubate at room temperature for 2-3 minutes;
2.5 Centrifuge 12000g at 4 ° C for 15 minutes;
Attention: The lysate is divided into a red phenol BCP layer (chloroform layer), an intermediate layer, and a colorless aqueous phase. RNA is completely retained in the aqueous phase. The upper water phase accounts for approximately 50% of the total volume;
2.6 Tilt the test tube 45 degrees., Be careful to extract the upper layer solution and do not disturb the middle layer and phenol layer;
2.7 Transfer the upper solution to a new test tube;
3 RNA precipitation
Take appropriate preventive measures to avoid RNase contamination.
3.1 (Optional) When precipitating RNA from small samples (<10 ° cells or<10mg tissue), 5-10 μ g of RNase free glycogen should be added to the aqueous phase; Attention: Glycogen co precipitates with RNA, and when its concentration is ≤ 4mg/mL, it does not inhibit the synthesis of the first strand or the PCR reaction.
3.2 Add 0.5mL of isopropanol to the aqueous phase obtained after homogenizing 1mL of Trizol;
3.3 Incubate at room temperature for 10 minutes;
3.4 Centrifuge at 12000g for 10 minutes at 4 ° C;
Note: Before centrifugation, RNA is not visible. After centrifugation, gel like precipitates form at the bottom and side walls of the test tube.
4 RNA washing
4.1 Remove the supernatant from the test tube, leaving only RNA precipitate;
4.2 Wash the precipitate with 1mL 75% ethanol (starting from the precipitate obtained from 1mL Trizol);
4.3 Briefly vortex, then centrifuge at 7500g for 5 minutes at 4 ° C to obtain the supernatant;
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