RNA quality control is necessary in molecular biology to ensure the success of sensitive downstream applications, such as quantitative polymerase chain reaction (qPCR) and next-generation sequencing. These techniques rely on high-quality input material for accurate results. Spectrophotometry is the most common and rapid method used to assess both the concentration and purity of an RNA sample. This technique measures the amount of ultraviolet light absorbed, providing a purity profile based on specific ratios. The 260/230 ratio measures chemical cleanliness, indicating residual organic compounds that could inhibit subsequent enzymatic reactions.
Understanding the Wavelengths
The 260/230 ratio is calculated by comparing the absorbance reading at 260 nanometers (nm) to the absorbance reading at 230 nm. Nucleic acids, including RNA, DNA, and free nucleotides, absorb ultraviolet light most efficiently at 260 nm due to the structure of their aromatic bases. This 260 nm value forms the numerator of the purity ratio, representing the concentration of the target molecule.
The absorbance reading at 230 nm primarily measures chemical contamination from reagents used during RNA extraction. Many common compounds absorb strongly in this range. Chaotropic salts, such as guanidinium thiocyanate, used to lyse cells and stabilize RNA, are major culprits that elevate the 230 nm reading. Residual phenol, ethanol, and Trizol components are other organic contaminants that absorb light at this wavelength.
When contaminant concentration is high, the absorbance at 230 nm increases significantly. This increase in the denominator drives the final 260/230 ratio downward. A low 260/230 ratio signals a chemically impure sample, suggesting residual extraction reagents remain in the final RNA preparation.
Defining the Target Purity Range
For RNA suitable for most sensitive molecular applications, the 260/230 ratio should ideally fall within the range of 2.0 to 2.2. A ratio in this window indicates the RNA is relatively free from organic and chaotropic salt contamination, which inhibit enzymes used in downstream assays. This benchmark is widely accepted as the standard for a clean sample.
A ratio at 2.0 or slightly above suggests that the contribution of contaminating substances at 230 nm is minimal compared to the RNA signal at 260 nm. While 2.0 to 2.2 is the optimal range, many laboratories accept samples with a ratio above 1.8, especially for less sensitive applications. A ratio slightly higher than 2.2 is usually not a concern and suggests a very clean sample, though it can occasionally point toward an issue with the blank solution.
Troubleshooting Contamination
A 260/230 ratio below 1.8 indicates a need for further cleanup, as contaminants can interfere with enzymatic reactions. The most common cause of a low ratio is the carryover of guanidinium salts, used to denature proteins and maintain RNA stability. Residual phenol, a component of Trizol-based extractions, is another frequent contaminant that absorbs strongly at this wavelength.
For RNA purified using silica-column-based kits, a low ratio often means the wash steps were insufficient to remove the chaotropic salts. The most direct corrective action is to perform one or two additional wash steps using the manufacturer’s recommended wash buffer, typically 70–80% ethanol, to desalt the column. It is important to ensure the column is completely dry before elution, as residual ethanol carryover can also contribute to a low ratio.
When dealing with a low ratio from a Trizol-based extraction, the RNA pellet can be re-purified through an ethanol precipitation protocol. This involves re-dissolving the RNA pellet and washing it thoroughly with cold, 70% ethanol to remove residual phenol and salts. For problematic samples, or complex samples (e.g., those from plants containing carbohydrates), a commercial RNA clean-up kit can be used. This kit binds the RNA to a fresh column, separating it from soluble contaminants. The effectiveness of any cleanup step must be verified by re-measuring the 260/230 ratio before proceeding to enzyme-dependent experiments.