Ribonucleic acid (RNA) is a fundamental molecule in all living organisms, playing diverse roles in gene expression and regulation. Researchers frequently isolate RNA from biological samples for molecular biology applications. The success of these experiments depends on the quality and purity of the RNA sample.
Understanding RNA Purity Metrics
Scientists often use spectrophotometry to assess the concentration and purity of isolated RNA samples. This technique measures the amount of light absorbed by a sample at specific wavelengths. The 260/280 ratio indicates protein contamination, as proteins absorb light strongly at 280 nanometers (nm).
The 260/230 ratio assesses the presence of organic contaminants and chaotropic salts. Nucleic acids, including RNA, absorb light maximally at 260 nm. Common contaminants found in RNA extractions absorb light at or near 230 nm. These include guanidine salts (used in extraction buffers), phenol, carbohydrates, ethanol carryover, and EDTA.
The Significance of the 260/230 Ratio
The 260/230 ratio provides important information because contaminants absorbing at 230 nm can interfere with downstream enzymatic reactions. These substances often inhibit enzyme activity, making the RNA sample unsuitable for molecular biology techniques. A compromised purity ratio can render the sample unusable, even if the RNA concentration appears sufficient.
For example, guanidine salts can inhibit reverse transcriptase, an enzyme essential for converting RNA into complementary DNA (cDNA) in applications like reverse transcription quantitative PCR (RT-qPCR). This inhibition can lead to reduced cDNA yield, inaccurate gene expression quantification, or reaction failure. Impurities can also affect DNA polymerase efficiency in subsequent PCR amplification.
In RNA sequencing, a low 260/230 ratio can result in inefficient library preparation. This may lead to low sequencing yields, biased RNA molecule representation, or sequencing run failure. Maintaining a high 260/230 ratio is important for reliable and reproducible results across various molecular biology experiments.
Interpreting Your 260/230 Ratio
For a pure RNA sample, the generally accepted range for the 260/230 ratio is approximately 2.0 to 2.2. This optimal range indicates minimal contamination from substances that absorb at 230 nm. Slight variations in this ideal range can occur depending on the RNA extraction method or intended downstream application.
A 260/230 ratio significantly below this range, such as values closer to 1.0 or lower, indicates substantial contamination. Common culprits include carryover of guanidine salts, residual phenol, ethanol, carbohydrates, and EDTA. These contaminants interfere with enzymatic reactions and accurate spectrophotometric readings.
While less common, a 260/230 ratio significantly above the ideal range might also occur. This could suggest issues such as an acidic pH of the elution buffer or an inappropriately diluted blank solution during spectrophotometric measurement. For a comprehensive assessment of RNA purity, both the 260/280 and 260/230 ratios should be considered.
Practical Approaches to Optimizing Your 260/230 Ratio
Achieving an optimal 260/230 ratio begins with attention to detail during RNA extraction. Thorough washing steps are important to effectively remove residual contaminants. Efficient ethanol washes, especially during column-based purification, help eliminate chaotropic salts and other organic solvents.
Careful aspiration prevents carryover of supernatant or residual ethanol from wash buffers into the final RNA elution. Any remaining liquid from previous steps can introduce impurities that depress the 260/230 ratio. Using high-quality, fresh reagents and nuclease-free water throughout the extraction minimizes new contaminants.
Avoid overloading extraction columns or exceeding recommended sample input for RNA purification kits. Overloading can lead to inefficient washing and incomplete removal of impurities, resulting in a lower purity ratio. Adhering to manufacturer’s guidelines for sample input is important.
If the 260/230 ratio remains low after initial extraction, re-purification of the RNA sample may be necessary. This can be accomplished using specialized spin columns for RNA cleanup or through precipitation methods like ethanol precipitation. Before re-measuring, verify the blank solution used for spectrophotometry is appropriate for the elution buffer.