Concentrating ribonucleic acid (RNA) is a fundamental laboratory technique that reduces solvent volume while increasing solute amount. This process is performed after initial purification to prepare samples for sensitive downstream applications. Techniques like quantitative polymerase chain reaction (qPCR) and next-generation sequencing require a precise, high concentration of input material. Successful concentration maximizes experimental effectiveness and reliability.
Concentration Through Alcohol Precipitation
The classic method for concentrating RNA relies on alcohol precipitation. RNA is hydrophilic due to negatively charged phosphate groups, allowing it to dissolve easily in water. To precipitate RNA, the negative charges must be neutralized, and the solvent’s polarity drastically reduced. This is achieved by adding a salt, such as sodium acetate or lithium chloride, and a high concentration of alcohol, typically ethanol or isopropanol.
The positively charged ions from the salt, such as sodium or lithium, interact with and neutralize the negative phosphate charges on the RNA molecule. Adding a large volume of alcohol, typically two to three volumes of 95% to 100% ethanol, reduces the overall dielectric constant of the solution. This solvent change makes the neutralized RNA molecules insoluble, causing them to aggregate and fall out of solution. Incubation, often at cold temperatures like -20°C, can improve aggregation efficiency, especially for low-concentration samples.
A carrier molecule like glycogen or linear polyacrylamide may be added to increase the mass of the resulting pellet, making recovery easier. The precipitated RNA is then collected by high-speed centrifugation, which forces the insoluble material to the bottom of the tube. After the supernatant is removed, the pellet is washed with a cold 70% ethanol solution. This wash step removes residual salts and impurities without redissolving the RNA, improving the purity of the final concentrated sample.
Utilizing Filtration and Spin Columns
A modern and rapid approach uses commercial kits based on filtration and spin column technology. This method utilizes a silica-based membrane housed within a small column for selective binding and purification of the RNA. The process begins by mixing the sample with a specialized lysis and binding buffer containing chaotropic salts, such as guanidinium isothiocyanate.
Chaotropic salts disrupt the structure of water and other macromolecules, facilitating the binding of RNA to the silica membrane. Under these high-salt conditions, the RNA molecule’s phosphate backbone interacts with the silica surface through various forces, including hydrogen bonds and electrostatic forces. The sample is loaded onto the spin column, and centrifugation forces the liquid through the membrane, leaving the RNA selectively bound.
Following the binding step, wash buffers containing alcohol are passed through the column to remove contaminants, including proteins and residual salts. Because the RNA remains tightly bound to the silica, impurities are effectively washed away with the flow-through. This solid-phase extraction significantly reduces handling time and the complexity of the purification process compared to precipitation.
The final step is elution, which involves adding a minimal volume of a low-ionic strength buffer, such as nuclease-free water or a weak Tris-EDTA solution, directly to the membrane. The change in ionic conditions disrupts the interaction between the RNA and the silica, allowing the now-concentrated RNA to be released into the small volume of eluent. This streamlined process results in high-purity, concentrated RNA.
Reducing Sample Volume with Vacuum Evaporation
Another physical method for concentrating RNA involves using a vacuum concentrator, often referred to as a centrifugal vacuum evaporator. This specialized instrument combines centrifugation, vacuum pressure, and sometimes low heat to rapidly remove the solvent from the sample tube. Reducing the atmospheric pressure lowers the boiling point of the solvent, allowing evaporation to occur quickly at reduced temperatures.
The centrifugal force applied during the process prevents the sample from “bumping” or boiling over, safeguarding against sample loss and cross-contamination. Since RNA is susceptible to degradation from heat, the vacuum environment permits solvent removal at temperatures that will not compromise sample integrity. For aqueous solutions, the evaporation process can cause the sample temperature to drop rapidly, requiring careful control to prevent unwanted freezing.
This technique is frequently used to concentrate RNA after alcohol precipitation or when the sample requires a smaller final volume. Once the solvent has been evaporated, the RNA is left as a dried or highly concentrated pellet at the bottom of the tube. The concentrated material must then be resuspended in a precise, minimal volume of an appropriate buffer to achieve the desired final concentration.
Confirming Concentration and Purity
After employing any concentration method, the final step involves confirming both the concentration and the purity of the resulting RNA sample. Accurate quantification is necessary because many molecular applications require a specific input amount for optimal performance. Two primary methods are used for this quality control: spectrophotometry and fluorescence-based assays.
Spectrophotometry measures the absorption of ultraviolet light at 260 nanometers (A260), which is directly proportional to the nucleic acid concentration. This method also provides purity checks by measuring absorbance ratios. The A260/A280 ratio indicates protein contamination (ideal value near 2.0), while the A260/A230 ratio assesses residual organic solvents or chaotropic salts (values typically above 1.8 indicate good purity).
While spectrophotometry is fast, it measures all nucleic acids present, including contaminating DNA, and can be easily influenced by impurities that absorb light. Fluorescence-based assays, such as those using the Qubit system, offer higher sensitivity and greater specificity. These assays use fluorescent dyes that only bind to the target RNA molecule, providing accurate quantification even in the presence of contaminating DNA or proteins.