RNA purification is the fundamental process of isolating high-quality, non-degraded ribonucleic acid (RNA) from a biological sample. The integrity and purity of the isolated RNA directly influence the accuracy of subsequent experiments. Researchers rely on purified RNA for downstream applications, such as quantifying gene expression through quantitative polymerase chain reaction, performing next-generation sequencing, and analyzing complex cellular pathways. The success of these techniques requires an intact and contaminant-free RNA sample.
Preventing Degradation Before Extraction
The moment a biological sample is collected, its RNA is under immediate threat from ribonucleases (RNases). These enzymes are ubiquitous, originating both from within the sample and from the surrounding environment, including human skin and dust. To counteract this, every step of sample handling must be performed in a strictly RNase-free environment. Equipment and solutions are often treated with chemicals like diethyl pyrocarbonate (DEPC), and laboratory surfaces are decontaminated with commercial RNase-inactivating solutions.
Immediate sample stabilization is necessary to halt endogenous RNase activity before purification begins. One common technique is flash-freezing the sample in liquid nitrogen, which instantly freezes cellular components and arrests enzymatic processes. Alternatively, chemical stabilization buffers such as RNAlater can preserve the tissue’s RNA integrity at room temperature for a short period. Once stabilized, the sample must be thoroughly homogenized to ensure complete cell lysis and release of the RNA into the extraction buffer.
Effective homogenization is achieved through mechanical methods, such as grinding frozen tissue with a mortar and pestle, or using bead mill homogenizers. This disruption must be paired with the immediate application of a lysis buffer containing protein denaturants. Chaotropic agents, such as guanidinium thiocyanate, dissolve cell membranes and rapidly inactivate RNase enzymes. Complete cell disruption is necessary to maximize the RNA yield and ensure all RNA molecules are exposed to the protective lysis solution.
Chemical Separation Using Liquid-Phase Methods
One established method for purifying RNA is the liquid-phase separation technique, often called Acid Guanidinium Thiocyanate-Phenol-Chloroform extraction. This method relies on the differential solubility of nucleic acids and proteins in specific organic solvents at an acidic pH. The initial step involves lysing the sample with a solution containing guanidinium thiocyanate, which denatures proteins and solubilizes the cellular contents.
The addition of an acidic phenol solution and chloroform causes the mixture to separate into three distinct layers upon centrifugation. The acidic pH ensures that DNA and most cellular proteins are partitioned into the lower, denser organic phase. The middle layer, or interphase, primarily contains denatured proteins and cellular debris. The target RNA molecules remain dissolved in the top aqueous phase due to their chemical properties under these acidic conditions.
The aqueous phase containing the purified RNA is carefully transferred to a new tube, avoiding contamination from the interphase or organic layer. RNA is then recovered through alcohol precipitation, typically by adding isopropanol. The alcohol reduces the solubility of the RNA, causing it to precipitate into a pellet upon centrifugation. This pellet is then washed with a high-concentration ethanol solution to remove residual salts and organic contaminants, yielding a pure RNA product.
Isolation Through Solid-Phase Techniques
Solid-phase extraction, commonly utilizing spin columns, offers a faster and less hazardous alternative to liquid-phase separation by eliminating organic solvents. This method relies on the principle of selective adsorption, where nucleic acids bind to a silica-based membrane under high-salt conditions. The process begins with sample lysis, using a buffer containing a chaotropic salt, such as guanidinium hydrochloride, to break open cells and inactivate RNases.
The lysate is applied to a column containing the silica membrane, and centrifugation forces the solution through the filter. The high concentration of chaotropic salts promotes the binding of the negatively charged RNA backbone to the silica surface. Contaminants, including proteins, lipids, and cellular debris, pass through the membrane into the collection tube, leaving the RNA selectively bound.
A series of wash steps is performed next, using ethanol-based buffers that maintain RNA binding conditions while removing residual salts and impurities. These wash solutions ensure a high-purity product by stripping away weakly bound contaminants that might interfere with downstream enzymatic reactions. The final step is elution, where a low-ionic strength solution, such as RNase-free water or a specialized buffer, is applied to the membrane. This low-salt environment rehydrates the silica, releasing the bound RNA molecules, which are then collected.
Assessing Quality and Ensuring Stability
After purification, quality control measures are necessary to confirm that the isolated RNA is suitable for sensitive downstream applications. Quantification is a primary step, often performed using spectrophotometry, such as a Nanodrop device, which measures concentration based on ultraviolet light absorbance at 260 nanometers. A more specific method is fluorometry, using instruments like the Qubit, which employs fluorescent dyes that only bind to RNA, providing a more accurate concentration measurement by excluding contaminating DNA.
Purity assessment relies on spectrophotometric ratios, with the A260/280 ratio indicating residual protein contamination; an optimal value for pure RNA is approximately 2.0. The A260/230 ratio measures contamination by organic compounds and chaotropic salts, with values ideally falling between 2.0 and 2.2. Deviations from these ranges suggest the presence of carryover contaminants that could inhibit enzymes in subsequent experiments.
RNA integrity is assessed to confirm that the molecules have not been degraded during purification. This is most accurately measured using automated electrophoresis systems, such as a Bioanalyzer, which generates an RNA Integrity Number (RIN). This score ranges from 1 (completely degraded) to 10 (intact), providing an objective measure of quality by analyzing the ribosomal RNA peaks. For long-term preservation, purified RNA must be stored in small, single-use aliquots at ultra-low temperatures, ideally between -70°C and -80°C, to minimize degradation from repeated freeze-thaw cycles.