Ribonucleic acid (RNA) serves as the messenger that translates genetic instructions stored in DNA into the functional components of a cell. Studying RNA provides a real-time snapshot of the genes actively working within an organism or tissue. Isolating this delicate molecule from the dense mix of cellular components, such as proteins, DNA, and lipids, is challenging due to its high susceptibility to enzymatic degradation. The widely adopted TRIzol method, based on guanidinium thiocyanate and phenol, offers a highly effective technique for extracting high-quality, intact RNA. This chemical approach is a reliable standard for ensuring the harvested RNA is suitable for sensitive downstream molecular analyses, such as sequencing or gene expression studies.
The Chemical Action of the Lysis Reagent
The process begins by introducing the biological sample to a powerful lysis reagent, a monophasic solution containing phenol and guanidinium thiocyanate. Guanidinium thiocyanate is a strong chaotropic agent, meaning it disrupts the ordered structure of water molecules and subsequently interferes with non-covalent molecular forces within the cell. This disruption causes cell walls and membranes to instantly dissolve, rapidly releasing the cell contents into the solution.
The reagent must also protect the released RNA from rapid degradation. Guanidinium thiocyanate powerfully denatures all proteins, including RNases—enzymes that break down RNA molecules. The swift inactivation of these destructive enzymes is critical for preserving the RNA’s integrity immediately upon cell rupture.
Phenol works with the guanidinium salt to maintain the solution’s acidic pH, typically around 4.0. This low pH dictates how nucleic acids and proteins behave in subsequent separation steps. Under acidic conditions, the reagent ensures that most lipids and hydrophobic molecules are efficiently solubilized within the phenol component, allowing for the selective partitioning of RNA from other cellular debris.
Leveraging Differential Phase Separation
Following lysis, differential phase separation is initiated by adding chloroform, or a similar phase-separating agent, to the mixture. Chloroform is immiscible with the aqueous components. Due to its density, centrifugation drives the separation of the mixture into three distinct layers. The high-speed spinning physically separates components based on their density, polarity, and solubility characteristics.
The separated sample displays three layers. The dense, dark red lower layer is the organic phase, composed primarily of phenol and chloroform. Floating above this is the light, clear, water-based upper layer, known as the aqueous phase. Between these two liquids lies the thin, whitish interphase, which accumulates denatured proteins and large DNA fragments.
The acidic pH established during lysis determines where cellular macromolecules partition. In this low pH environment, DNA and proteins become protonated, losing their negative charge. This makes them more soluble in the organic phenol layer or causes them to precipitate at the interphase. The chemical affinity of proteins for phenol is maximized under these conditions, sequestering them away from the RNA.
Conversely, highly polar RNA molecules retain their strong negative charge even in the acidic environment, preventing them from migrating into the organic phase. This property ensures that the RNA remains selectively partitioned and fully dissolved within the upper aqueous phase, away from contaminants. Researchers carefully aspirate the upper, RNA-containing aqueous layer, typically taking about 60-70% of the volume, without disturbing the lower organic phase or the interphase. This isolation step prepares the sample for final purification.
Alcohol Precipitation and Final RNA Recovery
The collected aqueous phase contains purified RNA but still holds salts and residual chemical contaminants. To isolate the RNA, an equal volume of cold isopropanol is added, initiating alcohol precipitation. The alcohol drastically reduces the solubility of the negatively charged RNA molecules by altering the dielectric constant and disrupting the hydration shell.
Isopropanol changes the solution’s polarity, weakening the interactions between the RNA and water molecules that keep it dissolved. The negatively charged phosphate backbone of the RNA then aggregates and precipitates out of the solution, especially when cooled. High-speed centrifugation consolidates the precipitated RNA into a small, often translucent pellet at the bottom of the tube.
After pellet formation, the supernatant is discarded, and the pellet is washed using 75% ethanol. The ethanol wash removes residual contaminants, such as traces of guanidinium thiocyanate, phenol, or salts, which could interfere with later experiments. The 75% ethanol concentration ensures the RNA pellet remains insoluble while soluble contaminants are washed away.
The remaining liquid is removed after the final ethanol wash, and the RNA pellet is allowed to air-dry briefly. This ensures all traces of volatile alcohol are evaporated, which is important for downstream enzyme activity. The final step involves resuspending the purified RNA in a small volume of RNase-free water or a specialized storage buffer, resulting in a concentrated solution ready for use or preservation.
Quality Assessment and Storage of Extracted RNA
After RNA recovery, researchers must confirm the extraction success by assessing the quantity and purity of the final product. Spectrophotometry is the standard initial method used to determine RNA concentration by measuring absorbance at 260 nanometers (A260). Purity is simultaneously assessed by calculating specific absorbance ratios, which indicate the presence of common contaminants.
The A260/A280 ratio identifies protein contamination, as proteins and phenol absorb strongly at 280 nm; an ideal ratio for pure RNA is approximately 2.0. The A260/A230 ratio assesses contamination from guanidinium thiocyanate and carbohydrates, which absorb at 230 nm. A value typically falling between 2.0 and 2.2 indicates high purity. Ratios significantly below these values suggest insufficient wash steps and remaining contaminants.
The integrity of the RNA is evaluated using techniques such as agarose gel electrophoresis or specialized capillary electrophoresis instruments. These methods visualize the ribosomal RNA bands, which should appear sharp and distinct, providing an RNA Integrity Number (RIN). A high RIN value confirms the RNA has not been significantly degraded by RNases during extraction.
Proper storage is necessary to maintain the sample’s integrity. Purified RNA is chemically stable in RNase-free water but remains susceptible to degradation. For short-term use, it can be kept at -20°C. For long-term archiving, the RNA must be stored at ultra-low temperatures, typically -80°C, to halt all enzymatic and chemical breakdown processes.