Calculation of A260/A280 Ratio for Nucleic Acid Purity

The A260/A280 ratio is crucial for evaluating nucleic acid purity in molecular biology laboratories.
This guide explains the ratio’s calculation, offering formulas, reference tables, examples, and expert interpretations.

Common A260/A280 Ratio Values for Nucleic Acids

The table below displays the most commonly observed A260/A280 ratios for DNA and RNA samples along with their purity implications. These reference values are derived from empirical data in accordance with the current laboratory best practices.

Note: Consistent results require clean cuvettes, calibrated spectrophotometers, and proper blanking with the elution buffer.

Formulas Used for A260/A280 Ratio Calculation

1. Core Equation

The absorbance ratio is calculated using:

Where:

Instrumental Considerations:

• Measurements are taken using a UV-Vis spectrophotometer or microvolume platforms like NanoDrop™.
• Use quartz cuvettes for accuracy if not using microvolume tech.

2. Nucleic Acid Concentration Formulas

For Double-Stranded DNA (dsDNA):

For RNA:

For Single-Stranded DNA (ssDNA):

For Oligonucleotides:

Ensure dilution factor is included if applicable:

Explanation of Key Variables

Common Reference Standards and Normative Sources

The purity thresholds and interpretation of the A260/A280 ratio are supported by:

• MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments
• Thermo Fisher Scientific NanoDrop™ Protocols
• NIH Research Guidelines: Recommended A260/A280 purity metrics for RNA integrity and DNA prep.
• ISO 20387:2018 (Biotechnology – Biobanking standards)

Real-World Example #1: DNA Extraction from Blood

Scenario: A researcher extracts genomic DNA from peripheral blood using a silica column protocol and measures:

Step-by-step calculation:

1. Calculate A260/A280:

1. Concentration:

3. Interpretation:

• The A260/A280 ratio of 1.83 is close to the ideal 1.8, indicating pure DNA.
• Concentration is within usable range for PCR and gel electrophoresis.

4. Action:

• Sample is considered acceptable for downstream genotyping and PCR protocols.

Real-World Example #2: RNA Extraction from Plant Tissue

Scenario: A molecular biologist extracts total RNA from Arabidopsis thaliana leaves using TRIzol™ reagent and measures:

Step-by-step calculation:

1. Calculate A260/A280:

2. RNA Concentration (undiluted):

3. Adjusted for Dilution:

4. Interpretation:

• A260/A280 of 2.10 suggests high-purity RNA, slightly above ideal (2.0), but acceptable.
• Could indicate traces of phenol, common in TRIzol protocols.
• The high concentration is optimal for reverse transcription or RNA-seq.

5. Action:

• Sample may be treated with an additional ethanol precipitation step if downstream sensitivity to phenol is a concern.

Best Practices to Ensure Reliable A260/A280 Measurements

Accurate measurement of A260/A280 ratios is contingent on rigorous lab technique. Follow these guidelines:

Do:

• Use RNAse/DNAse-free consumables
• Blank the instrument with the same buffer used in elution
• Measure triplicates and take average
• Keep samples free from bubbles, fingerprints, or residual solvents

Avoid:

• Measuring too diluted samples (A260 < 0.1) – signal-to-noise is too low
• Using plastic cuvettes for UV – they absorb at 260 nm
• Failing to account for dilution factor – leads to underestimation

Factors Affecting the A260/A280 Ratio

Interpreting Unusual A260/A280 Ratios

Sometimes ratios fall outside the expected ranges, and understanding their meaning is critical.

Integration into Modern Molecular Workflows

The A260/A280 ratio is not a standalone indicator. Combine it with:

• A260/A230 ratio (ideal ~2.0–2.2): detects carbohydrate, phenol, or guanidine contamination
• RNA Integrity Number (RIN) from Bioanalyzer: ensures transcript quality
• Fluorometric methods (e.g., Qubit™): provide accurate quantification

For clinical labs and biobanks, compliance with ISO 20387:2018 and OECD Guidelines recommends dual metrics: A260/A280 and integrity assays for nucleic acid quality assurance.

Useful Online Resources

• Thermo Fisher NanoDrop Applications
• QIAGEN Nucleic Acid Purity Guide
• PubMed Central: Quantitation of Nucleic Acids
• OECD Biobanking Guidelines

Summary Table: A260/A280 Ideal Values by Application