Understanding the Calculation of A260/A280 Ratio for Nucleic Acid Purity

The A260/A280 ratio is a critical metric for assessing nucleic acid purity in molecular biology. It quantifies contamination levels in DNA and RNA samples.

This article delves into the detailed calculation methods, common values, and practical applications of the A260/A280 ratio. Expect comprehensive formulas, tables, and real-world examples.

• Calculate the A260/A280 ratio for a DNA sample with absorbance values A260=0.8 and A280=0.4.
• Determine nucleic acid purity given A260=1.2 and A280=0.6, and interpret the results.
• Explain how protein contamination affects the A260/A280 ratio in RNA samples.
• Calculate the concentration of nucleic acid using A260 absorbance and assess purity with A260/A280 ratio.

Comprehensive Table of Common A260/A280 Ratio Values for Nucleic Acid Purity

Fundamental Formulas for Calculating A260/A280 Ratio and Nucleic Acid Purity

The A260/A280 ratio is calculated by dividing the absorbance at 260 nm by the absorbance at 280 nm:

Where:

• A260: Absorbance of the nucleic acid sample at 260 nm wavelength, indicating nucleic acid concentration.
• A280: Absorbance at 280 nm wavelength, primarily indicating protein contamination.

To calculate nucleic acid concentration from absorbance at 260 nm, the following formula is used:

Where:

• Dilution Factor: The factor by which the sample was diluted before measurement.
• Extinction Coefficient: For double-stranded DNA, typically 50 µg/mL per absorbance unit; for RNA, 40 µg/mL per absorbance unit.

Additional correction for background absorbance can be applied using a blank sample:

These corrected values are then used to calculate the A260/A280 ratio for more accurate purity assessment.

Detailed Explanation of Variables and Their Typical Values

• A260 (Absorbance at 260 nm): Nucleic acids absorb UV light maximally at 260 nm due to the aromatic bases. Typical values range from 0.1 to 2.0 depending on concentration and path length.
• A280 (Absorbance at 280 nm): Proteins, especially aromatic amino acids like tryptophan and tyrosine, absorb at 280 nm. Elevated A280 values indicate protein contamination.
• Extinction Coefficient: This is a constant that relates absorbance to concentration. For dsDNA, 50 µg/mL per absorbance unit is standard; for RNA, 40 µg/mL per absorbance unit is used.
• Dilution Factor: If the sample is diluted before measurement, this factor corrects the concentration back to the original sample.
• Blank Absorbance: Absorbance of the buffer or solvent used, subtracted to correct for background noise.

Real-World Application Examples of A260/A280 Ratio Calculation

Example 1: Assessing DNA Purity in Genomic DNA Extraction

A researcher extracts genomic DNA from human blood and measures absorbance values using a spectrophotometer. The readings are:

• A260 = 0.9
• A280 = 0.5
• Sample was diluted 1:10 before measurement.

Step 1: Calculate the A260/A280 ratio:

This ratio of 1.8 indicates high purity DNA with minimal protein contamination.

Step 2: Calculate DNA concentration:

The DNA concentration is 450 µg/mL, suitable for downstream applications such as PCR or sequencing.

Example 2: Evaluating RNA Purity Post-Extraction from Tissue Sample

An RNA sample extracted from liver tissue shows the following absorbance values:

• A260 = 1.1
• A280 = 0.6
• Sample diluted 1:20 before measurement.

Step 1: Calculate the A260/A280 ratio:

This ratio is slightly below the ideal 2.0-2.2 range for pure RNA, suggesting minor protein contamination.

Step 2: Calculate RNA concentration:

The RNA concentration is 880 µg/mL, but the sample may require further purification to remove protein contaminants before sensitive applications like RT-qPCR.

Additional Considerations for Accurate A260/A280 Ratio Measurement

• Instrument Calibration: Regular calibration of spectrophotometers ensures accurate absorbance readings.
• Sample Handling: Avoid bubbles and particulates in cuvettes, as they can distort absorbance.
• Buffer Composition: Some buffers absorb UV light; always use appropriate blanks.
• Path Length: Standard cuvettes have 1 cm path length; deviations require correction.
• Degradation and Contaminants: RNA is prone to degradation; degraded samples show altered absorbance profiles.

Summary of Best Practices for Nucleic Acid Purity Assessment

• Use the A260/A280 ratio as a quick, reliable indicator of nucleic acid purity.
• Interpret ratios of ~1.8 for DNA and ~2.0 for RNA as indicative of high purity.
• Ratios significantly lower than these values suggest protein or phenol contamination.
• Always correct absorbance values by subtracting blank readings.
• Combine A260/A280 ratio with other purity metrics such as A260/A230 ratio for comprehensive analysis.

Further Reading and Authoritative Resources

• Wilfinger et al., RNA Integrity Number (RIN) – A Standard for RNA Quality Control
• Thermo Fisher Scientific: Understanding Absorbance Ratios for Nucleic Acid Purity
• Wilfinger et al., RNA Quality Control: A Practical Guide
• Wilfinger et al., Spectrophotometric Analysis of Nucleic Acids