Steps to extract RNA

In the vast exploration of biological sciences, RNA extraction is undoubtedly a key step in unlocking the mysteries of life, and every link requires careful planning and execution. In order to efficiently and purely isolate valuable RNA molecules from cells, tissues or other biological samples, we must follow the following series of rigorous and meticulous operating procedures:

1. Experimental preparation

1. Prepare reagents and equipment: Prepare all reagents and equipment required for the experiment, such as centrifuge tubes, pipettes, cell scrapers, mortar, liquid nitrogen, RNA protectant, Trizol reagent, chloroform, isopropyl alcohol, 75% Ethanol, RNase-free water or buffer, etc.

2. Safety protection: Wear laboratory clothes, protective glasses and other necessary protective measures to ensure experimental safety.

3. Read the experimental steps: Read the experimental steps carefully and prepare a notebook to record the experimental process and results.

2. Sample collection and processing

1. Select samples: Select appropriate samples according to experimental requirements, such as cells, tissues, serum, urine, etc. Make sure the sample is representative and add RNA protectant immediately to prevent RNA degradation.

2. Sample preservation: Store the collected samples under low temperature conditions (such as liquid nitrogen or -80°C refrigerator) to maintain the integrity of the RNA.

3. Cell disruption

1. Mechanical disruption: Use mechanical methods such as high-speed centrifuge, ultrasonic device or liquid nitrogen freezing to disrupt the cells. For tissue samples, a pre-chilled mortar can be used to grind the sample into a powder.

2. Chemical disruption: Add an appropriate amount of cell lysis solution (such as Trizol reagent) to the cells or tissue samples, and pipet the sample repeatedly with a cell scraper until the cells are completely lysed.

4. RNA extraction

1. Stratification: Add an equal volume of chloroform to the cell lysis solution, mix thoroughly and centrifuge. After centrifugation, the sample will be divided into three layers: the upper layer is a colorless aqueous phase (mainly containing RNA), the middle layer is a layer containing DNA, and the bottom layer is an organic layer.

2. Transfer the aqueous phase: Carefully transfer the upper aqueous phase to a new centrifuge tube to avoid inhaling the middle layer and organic layer.

3. Precipitate RNA: Add an equal volume of isopropyl alcohol to the water phase, mix thoroughly and centrifuge to precipitate RNA to the bottom of the centrifuge tube.

4. Wash RNA: Carefully pour out the supernatant and wash the RNA pellet with 75% ethanol to remove isopropyl alcohol and other impurities.

5. Dry RNA: Dry the centrifuge tube in a ventilated place or absorb the remaining ethanol with absorbent paper. Be careful not to completely dry the RNA to avoid difficulty in dissolving.

5. Dissolve RNA

1. Dissolve RNA: Add an appropriate amount of RNase-free water or buffer to the centrifuge tube, and gently pipet to dissolve the RNA precipitate.

2. Heating and dissolving: Place the centrifuge tube in a 70°C water bath and heat it for a moment to completely dissolve the RNA.

6. RNA quantification and preservation

1. RNA quantification: Use UV absorption spectroscopy or fluorescence analyzer to determine the concentration and purity of RNA.

2. RNA preservation: Store RNA at a low temperature of -80°C, or add RNase inhibitors and other substances to avoid RNA degradation and contamination.

7. Precautions

1. Prevent RNase contamination: RNase (RNase) exists widely in the environment and can degrade RNA. Therefore, throughout the entire process of RNA extraction, RNase-free plastic products and glassware should be used, and new gloves should be replaced frequently to prevent RNase contamination.

2. Control experimental conditions: RNA is easily degraded under alkaline conditions, so RNA extraction should be carried out at a pH value lower than 7. At the same time, attention should be paid to maintaining low temperature conditions during the experiment to prevent RNA degradation.

3. Pay attention to operational details: During steps such as transferring the aqueous phase, precipitating RNA, and washing RNA, care should be taken to avoid loss of RNA or introduction of impurities.

This article only provides a basic operation of the RNA extraction process. For RNA extraction that is more professional, efficient and meets specific experimental needs, it is recommended that experimental operators refer to authoritative professional books, the latest research papers or official technical information. Because the specific goals of different experiments will directly lead to differences in adjusting the amount of reagents used, extraction temperature, lengthening or shortening of certain steps, etc.