Trizol method for RNA extraction

RNA extraction is a very important basic step in molecular biology research, and the extraction efficiency and purity directly affect the success of subsequent experiments. Trizol® Reagent is an efficient and reliable nucleic acid extraction reagent that provides a professional solution for researchers.

Trizol Features:

1. Efficient RNA extraction

Trizol® reagent is based on the classic acidic phenol-chloroform separation method for rapid extraction of high-purity RNA from a variety of samples such as cells, tissues, and plants. It can meet a variety of experimental needs, saving time and efficiency.

2. High purity

The extracted RNA has extremely high purity and integrity, with A260/280 values typically in the range of 1.8-2.0, which fully meets the requirements of molecular biology experiments.

3. Wide range of application

Sample type: Suitable for a variety of samples such as cells, tissues, bacteria, yeast, and viruses.

Downstream experiments: Extracted nucleic acids can be used directly for Northern, point hybridization, mRNA purification, in vitro translation, RNase protectionassay, cDNA cloning, and RT-PCR; It can also be used for gene expression chip analysis, high-throughput sequencing and other experiments with high requirements for RNA quality to ensure the accuracy and reliability of the results.

Reagents needed

Consumables needed

Steps:

1. Cell lysis

★ Tissue samples

Trizol dosage: Add 1 mL of Trizol per 50-100 mg of tissue, and the sample volume should not exceed 10% of the Trizol volume.

(1) Animal or plant tissues are cut into small pieces, ground in liquid nitrogen or homogenized with a homogenizer;

(2) Quickly transfer the ground tissue powder into a 2mL centrifuge tube filled with 1mL Trizol, quickly shake and mix on a vortex shaker, place on ice, and wait for all samples to be ground;

(3) The pyrolysis product should be a clear transparent viscous liquid. For tissue samples rich in protein, fat, or polysaccharide substances, such as muscle, adipose tissue, and plant nodules, where insoluble material remains after homogenization, centrifugation at 12,000 x g at 4°C for 10 minutes, and then pipetting the supernatant into a new centrifuge tube.

★ Cell samples

(1) Adherent cells: aspirate the culture medium, add 1mL of Trizol per 10cm² culture area (6-well plate single well or 35mm plate), pipette several times with a sampler to ensure complete lysis of the cells, and then transfer to a centrifuge tube;

Note: The amount of Trizol used should be determined by the surface volume of the dish, not by the number of cells. Insufficient amounts of Trizol may result in DNA contamination in the extracted RNA.

(2) Suspension cells: Collect cells by centrifugation, aspirate the liquid, and 10 × every 5-10⁶animal and plant or yeast cells, or every 1 × 10⁷Bacterial cells were added to 1 mL of Trizol, pipetted with a dispenser to allow them to be completely lysed, and then transferred to a centrifuge tube.

Note: Do not wash cells before adding Trizol to avoid RNA degradation. If necessary, a homogenizer can be used to lyse certain bacterial or yeast cells.

2. Liquid-phase separation

(1) The lysate is placed at room temperature for 5min to completely separate the nucleic acid-protein complex (note: the sample can be stored at -80°C for a long time at this time);

(2) Add 0.2mL chloroform to every 1mL of Trizol, close the cap tightly, shake vigorously for 15s, and place at room temperature for 2-3min;

(3) Centrifugation at 12,000xg at 4°C for 15min, the sample will be divided into three layers: orange lower organic phase, middle layer and colorless upper aqueous phase;

(4) Pipette the upper aqueous phase containing total RNA into a new centrifuge tube in a volume of 60% of the Trizol reagent used.

3. RNA recovery

(1) Add 0.5mL of isopropanol according to the initial dosage of 1mL of Trizol, mix it upside down several times, and place it at room temperature for 10min;

(2) Centrifuge at 12,000xg at 4°C for 10min, discard the supernatant, and the gelatinous RNA precipitate can be seen;

(3) Add 1mL of 75% ethanol according to the initial amount of 1mL of Trizol, mix it several times, and wash the precipitate;

(4) Centrifuge at 12,000xg at 4°C for 5min, and discard the supernatant;

(5) Invert at room temperature for 5-10min to dry or vacuum dry (do not use a vacuum drying centrifuge to avoid RNA being too dry and difficult to dissolve);

(6) Add an appropriate amount (such as 25uL) of DEPC-ddH₂O or TE buffer, pipetting several times with an injector to dissolve the RNA;

(7) RNA concentration, purity and integrity were determined by RNA electrophoresis and ultraviolet spectrophotometer;

(8) The obtained RNA should be used immediately or stored at -80 °C after appropriate aliquots to avoid repeated freezing and thawing.

In just a few steps, you can get high-quality RNA, which is simple and efficient. In addition to Trizol (abs60154), Absin also has Trizol (Total RNA Extraction Kit) (abs9331) which contains reagents for the entire RNA extraction process.

Absin provides antibodies, proteins, ELISA kits, cell culture, detection kits, and other research reagents. If you have any product needs, please contact us.

Absin Bioscience Inc. Email: worldwide@absin.cn

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