RNA Nanodrop 260/230: Interpreting Purity Ratios

By Dr. Zubair Khalid, DVM, MS, PhD · 2026-08-11

Introduction to RNA Nanodrop 260/230

What is the 260/230 ratio?

The 260/230 ratio is a spectrophotometric measurement that compares a sample's absorbance at 260 nanometers (nm) to its absorbance at 230 nm. In RNA quality assessment, this ratio serves as an indicator of chemical purity, specifically detecting contamination by organic compounds and chaotropic salts that absorb light in the 230 nm region. When you load a purified RNA sample into a NanoDrop spectrophotometer, the instrument measures how much ultraviolet (UV) light the sample absorbs at these two specific wavelengths and calculates the quotient.

A pure RNA sample typically yields a 260/230 ratio between 2.0 and 2.2. Values below this range suggest the presence of contaminants that absorb at 230 nm, while values significantly above 2.2 may indicate an error in measurement or an unusual buffer composition. The ratio is unitless—it is a quotient of two absorbance values—and therefore does not depend on sample concentration, provided the concentration falls within the linear detection range of the instrument.

The 260/230 ratio complements the more familiar Nanodrop 260/280 ratio, which assesses protein contamination. While the 260/280 ratio tells you about protein carryover, the 260/230 ratio provides information about a different class of contaminants, many of which are introduced during RNA isolation procedures using guanidine-based reagents.

Why RNA purity matters

RNA purity is not an abstract concern—it directly affects the reliability and interpretability of downstream applications. Impure RNA can produce misleading results in quantitative reverse transcription PCR (RT-qPCR), where contaminating compounds can inhibit reverse transcriptase and DNA polymerase enzymes. For example, guanidine thiocyanate, the active ingredient in many RNA lysis buffers such as TRIzol (Thermo Fisher) or TRI Reagent (Sigma-Aldrich), is a potent protein denaturant that irreversibly inhibits enzymatic reactions if carried over into the final RNA sample.

Similarly, phenol, used in organic phase separation during RNA extraction, can interfere with spectrophotometric quantification and inhibit downstream enzymatic steps. Even low concentrations of these contaminants—too low to detect by eye or by gel electrophoresis—can reduce the efficiency of cDNA synthesis, alter PCR amplification curves, and compromise microarray or next-generation sequencing (NGS) library preparation.

For undergraduate laboratory courses and research projects, understanding the 260/230 ratio helps you diagnose whether your RNA isolation protocol worked correctly and whether your sample is suitable for the intended application. A low 260/230 ratio does not necessarily mean your RNA is degraded, but it does mean your sample contains non-RNA material that may interfere with your experiments.

The Science Behind the 260/230 Ratio

Absorbance at 260 nm and 230 nm

Nucleic acids absorb UV light maximally at 260 nm due to the aromatic ring structures of the purine and pyrimidine bases. Adenine, guanine, cytosine, and uracil each contain conjugated double-bond systems that absorb light in this region. The molar extinction coefficient of RNA at 260 nm is approximately 40 ng·cm/µL (for a 1 cm path length), which is the basis for the standard conversion factor used by NanoDrop instruments to calculate RNA concentration from absorbance.

Absorbance at 230 nm, in contrast, is not primarily due to nucleic acids. Many organic compounds, including phenol, guanidine salts, and carbohydrates, have absorbance maxima near 230 nm. The peptide bonds of proteins also absorb in this region, though less strongly than at 280 nm where aromatic amino acids (tryptophan, tyrosine, and phenylalanine) dominate. The 230 nm wavelength was selected because it captures a broad range of chemical contaminants that commonly co-purify with nucleic acids during extraction procedures.

The ratio of absorbance at these two wavelengths provides a sensitive indicator of contamination because even small amounts of 230 nm-absorbing compounds disproportionately lower the ratio. This sensitivity arises because the ratio is a quotient—if the 260 nm absorbance is 0.5 and the 230 nm absorbance is 0.25, the ratio is 2.0. If contamination raises the 230 nm absorbance to 0.3, the ratio drops to 1.67, a 16.5% change that is easily detectable.

Contaminants that absorb at 230 nm

Several classes of compounds absorb significantly at 230 nm and are common contaminants in RNA preparations:

Guanidine salts: Guanidine thiocyanate and guanidine hydrochloride are chaotropic agents used to denature proteins and inactivate RNases during cell lysis. They have strong absorbance around 230 nm. Residual guanidine is the most common cause of low 260/230 ratios in RNA samples prepared with silica column kits or organic extraction methods.

Phenol: Used in the classic acid-phenol-chloroform RNA extraction method, phenol absorbs UV light across a broad range but has significant absorbance near 230 nm. Trace phenol carryover in the aqueous phase lowers the 260/230 ratio.

Carbohydrates: Polysaccharides and other sugars absorb at 230 nm. These are particularly problematic when isolating RNA from plant tissues, which are rich in carbohydrates, or from yeast and bacterial cultures with thick cell walls.

EDTA: Ethylenediaminetetraacetic acid, commonly included in TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH 8.0), absorbs at 230 nm. If RNA is eluted or resuspended in TE buffer rather than nuclease-free water, the EDTA contribution can lower the 260/230 ratio.

Residual ethanol: Ethanol itself has minimal absorbance at 230 nm, but ethanol precipitation can co-precipitate salts and other contaminants that do absorb at this wavelength. Incomplete removal of the ethanol supernatant can therefore indirectly lower the ratio.

Phenol red and other dyes: Some cell culture media contain phenol red, which absorbs in the 230 nm region. If RNA is isolated from cultured cells without adequate washing, residual dye can contaminate the final sample.

The table below summarizes common contaminants, their sources, and their primary absorbance characteristics:

How to Measure RNA Purity with a Nanodrop

Preparing the instrument

Before measuring your RNA sample, the NanoDrop instrument must be properly prepared to ensure accurate readings. The following steps apply to the NanoDrop One, NanoDrop 2000, and similar models. If you are using a different model, consult the Nanodrop Eight User Manual or Nanodrop Ultra User Manual for model-specific instructions.

1. Turn on the instrument and allow it to warm up for at least 5 minutes. The lamp needs time to stabilize for consistent readings.
2. Select the nucleic acid application from the home screen. Choose "RNA" as the sample type so the instrument applies the correct extinction coefficient (40 ng·cm/µL).
3. Clean the measurement pedestals. Using a lint-free laboratory wipe, gently clean both the upper and lower pedestals. If necessary, pipette 2-3 µL of nuclease-free water onto the lower pedestal, lower the arm, and lift it again to remove any dried residue. Wipe dry.
4. Blank the instrument. Pipette 1-2 µL of the same buffer used to elute or resuspend your RNA (typically nuclease-free water or 10 mM Tris-Cl, pH 8.0) onto the lower pedestal. Lower the arm and select "Blank." The instrument measures the blank solution and subtracts its absorbance from all subsequent sample readings. This step is critical—if you blank with water but your RNA is in TE buffer, the EDTA in the buffer will contribute to the 230 nm absorbance and artificially lower your 260/230 ratio.

Measuring the sample

Once the instrument is blanked:

1. Wipe the pedestals with a clean, lint-free wipe to remove the blank solution.
2. Pipette 1-2 µL of your RNA sample onto the lower pedestal. The volume must be sufficient to form a continuous liquid column between the upper and lower pedestals when the arm is lowered. For the NanoDrop 2000, 1 µL is typically sufficient; for the NanoDrop One, 2 µL is recommended.
3. Lower the sampling arm and select "Measure." The instrument will record the absorbance spectrum from 220 nm to 350 nm and calculate the RNA concentration, the 260/280 ratio, and the 260/230 ratio.
4. Record the results. Note the concentration, both ratios, and the shape of the absorbance spectrum. A clean RNA sample should show a smooth curve with a clear peak at 260 nm and a gradual decline toward 230 nm.
5. Wipe the pedestals immediately after measurement to prevent sample drying and crystallization on the optical surfaces.

Interpreting the output

The NanoDrop software displays several values on the results screen:

• Concentration: Reported in ng/µL, calculated from the absorbance at 260 nm using the equation: Concentration (ng/µL) = A260 × 40 (for RNA) × dilution factor.
• A260/A280: The ratio of absorbance at 260 nm to 280 nm. For pure RNA, this value should be approximately 2.0-2.2.
• A260/A230: The ratio of absorbance at 260 nm to 230 nm. For pure RNA, this value should be 2.0-2.2.
• Full spectrum: A graphical display of absorbance across the measured wavelength range. This is useful for diagnosing problems—for example, a peak at 270 nm suggests phenol contamination, while an elevated baseline across all wavelengths suggests particulate matter in the sample.

If your sample concentration is very low (below approximately 10 ng/µL), the absorbance values at both 260 nm and 230 nm will be small, and the ratios will be unreliable due to noise in the measurement. The NanoDrop One and similar instruments display a warning when the sample is too dilute for accurate ratio determination.

Interpreting 260/230 Values: What is Acceptable?

Expected values for pure RNA

For pure RNA in nuclease-free water or a low-salt buffer, the 260/230 ratio should fall between 2.0 and 2.2. Some sources cite a slightly wider acceptable range of 1.8 to 2.2, but values consistently below 2.0 warrant investigation.

The theoretical basis for this range lies in the absorbance properties of RNA itself. The ribose sugar backbone and the nitrogenous bases contribute to absorbance at both 260 nm and 230 nm, but the relative absorbance at these two wavelengths for pure RNA yields a ratio of approximately 2.0-2.2. This is slightly higher than the 260/280 ratio for pure RNA, which is typically 2.0-2.1.

It is important to note that the 260/230 ratio is more sensitive to buffer composition than the 260/280 ratio. If your RNA is dissolved in a buffer containing EDTA or other chelating agents, the 230 nm absorbance will increase, and the ratio will decrease even if the RNA itself is pure. For this reason, many protocols recommend eluting RNA in nuclease-free water rather than TE buffer when the sample will be used for spectrophotometric analysis.

What low 260/230 ratios mean

A 260/230 ratio below 1.8 indicates the presence of significant 230 nm-absorbing contaminants. The lower the ratio, the greater the contamination burden. As a rough guide:

• 1.8-2.0: Mild contamination. The sample may be usable for many applications, but caution is warranted for sensitive assays such as RT-qPCR or NGS library preparation.
• 1.5-1.8: Moderate contamination. The sample likely contains guanidine, phenol, or carbohydrate carryover. Additional purification is recommended.
• <1.5: Severe contamination. The sample is unlikely to perform well in enzymatic reactions. Consider re-purifying the RNA or re-isolating from the original source material.

A low 260/230 ratio does not necessarily mean your RNA is degraded. It is possible to have intact, high-quality RNA with a low 260/230 ratio if the isolation protocol left behind chemical contaminants. Conversely, it is possible to have degraded RNA with an acceptable 260/230 ratio if the degradation occurred without introducing 230 nm-absorbing compounds. The 260/230 ratio is a purity metric, not a quality metric—it tells you about contamination, not integrity.

Common Contaminants and Their Effect on 260/230

Guanidine salts

Guanidine thiocyanate (GuSCN) and guanidine hydrochloride (GuHCl) are chaotropic salts that denature proteins and inactivate RNases. They are components of virtually all commercial RNA lysis buffers, including:

• TRIzol/TRI Reagent: Contains guanidine thiocyanate and phenol
• Qiagen RLT buffer: Contains guanidine thiocyanate
• Promega SV RNA Lysis Buffer: Contains guanidine thiocyanate
• Zymo Research RNA Lysis Buffer: Contains guanidine thiocyanate

These compounds absorb strongly at 230 nm. If the washing steps in a silica column protocol are insufficient, or if the aqueous phase is not carefully separated during organic extraction, guanidine salts can carry over into the final RNA sample. The 260/230 ratio is particularly sensitive to guanidine contamination because these salts have a high molar absorptivity at 230 nm—even trace amounts can significantly lower the ratio.

To minimize guanidine carryover, follow the manufacturer's washing instructions precisely. Most silica column kits include an ethanol-based wash buffer (often called "RW" or "Wash Buffer") that removes guanidine salts. Ensure that you apply the correct volume and that you centrifuge for the recommended time. Some protocols include a "dry spin" step to remove residual ethanol, which also helps eliminate any remaining guanidine.

Phenol and other organics

Phenol is used in the acid-phenol-chloroform method for RNA extraction. In this method, cells are lysed in a solution containing phenol and guanidine thiocyanate, and chloroform is added to separate the mixture into an aqueous phase (containing RNA), an interphase (containing DNA and proteins), and an organic phase (containing phenol and lipids). If the aqueous phase is collected without adequate care, trace phenol can be carried over.

Phenol absorbs UV light with a peak around 270 nm and significant absorbance at 230 nm. Phenol contamination is often detectable by an abnormal absorbance spectrum—look for a shoulder or peak around 270 nm in the NanoDrop spectral display. The 260/280 ratio may also be elevated (above 2.2) in phenol-contaminated samples because phenol absorbs at 280 nm.

To remove phenol carryover, perform an additional chloroform extraction: add an equal volume of chloroform to the aqueous phase, mix, centrifuge, and collect the upper aqueous layer. Repeat if necessary. Ethanol precipitation can also help, as phenol remains in the supernatant while RNA precipitates.

Carbohydrates and other carryover

Carbohydrates are a common contaminant when isolating RNA from plant tissues, which can contain high levels of polysaccharides, or from yeast and bacteria with polysaccharide-rich cell walls. Carbohydrates absorb at 230 nm and can be difficult to remove once they co-precipitate with RNA.

Several strategies can reduce carbohydrate contamination:

• Use a lysis buffer with high salt concentration to precipitate polysaccharides before RNA binding.
• Include a high-salt precipitation step (e.g., 2 M lithium chloride) to selectively precipitate RNA while leaving carbohydrates in solution.
• Use a column-based kit designed for difficult tissues, such as those that include a specialized wash buffer for polysaccharide removal.

Other contaminants that can lower the 260/230 ratio include:

• EDTA from TE buffer (absorbance at 230 nm)
• Beta-mercaptoethanol (BME), used in some lysis buffers as an RNase inhibitor
• Dithiothreitol (DTT), another reducing agent used in RNA isolation
• Residual salts from precipitation procedures, such as sodium acetate or ammonium acetate

Troubleshooting Low 260/230 Ratios

Purification strategies

If your RNA sample has a low 260/230 ratio, several purification strategies can help, depending on the nature of the contaminant:

Ethanol precipitation: Add 0.1 volumes of 3 M sodium acetate (pH 5.2) and 2.5 volumes of ice-cold 100% ethanol to your RNA sample. Mix well and incubate at -20°C for at least 30 minutes (or -80°C for 15 minutes). Centrifuge at 12,000-15,000 × g for 15-30 minutes at 4°C. Carefully remove the supernatant, wash the pellet with 70% ethanol, centrifuge again, and air-dry the pellet for 5-10 minutes. Resuspend in nuclease-free water. This procedure removes guanidine salts, phenol, and many other small-molecule contaminants that remain in the supernatant.

Column-based cleanup: Commercial RNA cleanup kits, such as the Qiagen RNeasy MinElute Cleanup Kit or the Zymo Research RNA Clean & Concentrator Kit, can remove contaminants while concentrating your sample. These kits bind RNA to a silica membrane, wash away contaminants, and elute in a small volume. They are particularly effective for removing guanidine salts and residual phenol.

Size-exclusion spin columns: Columns such as the Bio-Rad Micro Bio-Spin P-30 or the GE Healthcare Illustra MicroSpin G-25 columns remove small molecules (<5 kDa) from RNA samples. These are useful for removing residual salts, nucleotides, and other small contaminants, but they will not remove large contaminants such as polysaccharides.

LiCl precipitation: For RNA samples contaminated with carbohydrates or proteins, lithium chloride precipitation can be effective. Add 0.3 volumes of 8 M LiCl to the RNA sample and incubate at -20°C for at least 1 hour. Centrifuge at 12,000 × g for 15 minutes at 4°C. The RNA pellet will contain less carbohydrate contamination than the original sample.

After any purification step, re-measure the 260/230 ratio to confirm improvement. Also re-measure the concentration, as purification steps inevitably result in some RNA loss.

When to re-isolate RNA

If purification steps do not improve the 260/230 ratio, or if the ratio remains below 1.5 after two rounds of cleanup, the most practical approach is to re-isolate RNA from the original source material. This is particularly true if:

• The RNA was isolated from a difficult tissue (plant, yeast, or bacterial samples with high carbohydrate content).
• The original isolation protocol was not followed precisely.
• The sample has been stored for an extended period, during which contaminants may have become more difficult to remove.
• The RNA concentration is too low to justify additional purification steps (purification always results in some loss).

When re-isolating, consider modifying the protocol to address the specific contamination issue. For example, if carbohydrate contamination was the problem, add a high-salt precipitation step or use a kit specifically designed for polysaccharide-rich tissues. If guanidine carryover was the issue, ensure that you are following the washing steps exactly and consider adding an extra wash.

Limitations of Nanodrop 260/230 for RNA Quality

Why 260/230 is not enough

The 260/230 ratio, like the 260/280 ratio, provides information about chemical purity but says nothing about RNA integrity. A sample can have perfect 260/230 and 260/280 ratios yet contain extensively degraded RNA. Conversely, a sample with slightly suboptimal ratios can contain intact, functional RNA.

The NanoDrop measures total absorbance at 260 nm, which includes contributions from all nucleic acids in the sample—RNA, DNA, and free nucleotides. It cannot distinguish between full-length RNA and fragmented RNA, nor can it distinguish RNA from DNA. If your RNA sample is contaminated with genomic DNA, the 260/230 ratio will not reveal this; you would need to check for DNA contamination using PCR with primers that amplify a genomic sequence, or by treating the sample with DNase and re-measuring.

Additionally, the NanoDrop measurement is affected by the pH and ionic strength of the sample buffer. RNA dissolved in unbuffered water can have a slightly lower pH due to dissolved CO₂, which can affect absorbance readings. The 260/230 ratio is particularly sensitive to pH changes because the absorbance of some contaminants varies with pH.

Complementary methods

To fully assess RNA quality, combine the NanoDrop ratios with integrity checks:

Agarose gel electrophoresis: Run 200-500 ng of RNA on a 1-1.5% denaturing agarose gel (or a native gel for a quick check). Stain with ethidium bromide or a safer dye such as SYBR Safe. Intact RNA shows two prominent bands corresponding to the 28S and 18S ribosomal RNA (in eukaryotic samples), with the 28S band approximately twice as intense as the 18S band. Degraded RNA appears as a smear with reduced or absent ribosomal bands.

Bioanalyzer or TapeStation: These microfluidic instruments provide a more quantitative assessment of RNA integrity. They generate an electropherogram and calculate an RNA Integrity Number (RIN) or RNA Quality Number (RQN) on a scale from 1 (fully degraded) to 10 (fully intact). A RIN of 7 or higher is generally considered acceptable for most downstream applications.

DNase treatment and PCR: To confirm the absence of genomic DNA contamination, treat an aliquot of your RNA sample with DNase I (RNase-free) and then perform PCR using primers specific to a genomic sequence. If no PCR product is amplified, the sample is free of DNA contamination.

Functional assay: The most definitive test of RNA quality is functional performance in a downstream application. If your RT-qPCR produces clean amplification curves with expected Ct values, your RNA is suitable for that application regardless of the spectrophotometric ratios.

Common Pitfalls in Measuring 260/230

Incorrect blanking

The most common error in NanoDrop measurements is improper blanking. The blank solution must match the sample buffer exactly. If you blank with nuclease-free water but your RNA is eluted in TE buffer, the EDTA in the TE buffer will absorb at 230 nm, and your 260/230 ratio will be artificially low. Conversely, if you blank with TE buffer but your RNA is in water, the instrument will subtract absorbance that is not present in your sample, potentially inflating the ratio.

Always elute or resuspend your RNA in the same buffer used for blanking. If you are unsure which buffer was used, re-suspend the RNA in a known buffer and re-measure.

Sample carryover

If the pedestals are not thoroughly cleaned between measurements, residual sample from a previous reading can contaminate the current measurement. This is particularly problematic when measuring samples with very different concentrations—a concentrated sample can leave behind enough material to significantly affect the next reading.

Always wipe the pedestals with a clean, lint-free wipe between measurements. If you suspect carryover, blank the instrument again before measuring your next sample. For samples with very high concentrations (>1000 ng/µL), consider measuring a blank (buffer only) between samples to verify that the pedestals are clean.

Dilution effects

The 260/230 ratio is concentration-dependent at low concentrations. When the RNA concentration is below approximately 10 ng/µL, the absorbance at 230 nm is very low (often below 0.02), and the instrument's noise floor can cause the ratio to fluctuate unpredictably. A sample with a true 260/230 ratio of 2.0 might read anywhere from 1.5 to 2.5 when measured at very low concentration.

If your sample is dilute, concentrate it by ethanol precipitation or use a microconcentrator before measuring. Alternatively, note that the ratio is unreliable and rely on other quality metrics.

Air bubbles

Air bubbles in the sample can scatter light and produce erratic absorbance readings. When pipetting the sample onto the pedestal, check that the liquid forms a continuous column without visible bubbles. If you see bubbles, wipe the pedestals and re-pipette.

Using the wrong application

The NanoDrop software has separate applications for RNA, DNA, and protein measurements. If you accidentally measure RNA using the DNA application, the instrument will apply the DNA extinction coefficient (50 ng·cm/µL) instead of the RNA coefficient (40 ng·cm/µL), and your concentration will be overestimated by 25%. The ratios will be unaffected, but the concentration will be wrong.

Dirty pedestals

Dried sample residue on the pedestals can scatter light and absorb at various wavelengths, producing distorted spectra and unreliable ratios. Clean the pedestals with a laboratory wipe and, if necessary, a small amount of 70% ethanol. Some models have a "clean" function that measures the pedestal without a sample to check for residue.

Practical Summary: Best Practices for RNA Quality Assessment

Quick checklist

Before measuring your RNA sample on a NanoDrop, run through this checklist:

1. Warm up the instrument for at least 5 minutes before use.
2. Clean the pedestals with a lint-free wipe. Check for visible residue.
3. Select the RNA application in the software.
4. Blank with the same buffer used to elute or resuspend your RNA.
5. Verify the blank spectrum is flat and near zero across the measured range.
6. Pipette 1-2 µL of sample onto the lower pedestal. Check for air bubbles.
7. Measure and record the concentration, 260/280, and 260/230 ratios.
8. Examine the spectrum for abnormal peaks or shoulders (e.g., at 270 nm for phenol).
9. Clean the pedestals immediately after measurement.
10. If the 260/230 ratio is low, consider purification or re-isolation.
11. Assess RNA integrity by gel electrophoresis or Bioanalyzer in addition to spectrophotometry.

Final recommendations

The 260/230 ratio is a useful but limited metric for RNA quality assessment. Use it as a screening tool, not as the sole determinant of RNA quality. A sample with a 260/230 ratio above 2.0 and a 260/280 ratio between 2.0 and 2.2 is likely free of significant chemical contamination, but you still need to check RNA integrity by gel electrophoresis or a microfluidic instrument.

When reporting RNA quality in your laboratory notebook or research paper, include the concentration, the 260/280 ratio, the 260/230 ratio, and the integrity assessment method. This provides a complete picture of RNA quality and allows others to evaluate whether your RNA was suitable for the downstream applications you performed.

For more detailed information on related measurements, see the Nanodrop A260/a280 and Nanodrop A260/a230 references. If you are using a specific NanoDrop model, consult the Nanodrop 8 User Manual or Nano-300 Nanodrop documentation for model-specific guidance.

Frequently Asked Questions

What is a good 260/230 ratio for RNA?

A good 260/230 ratio for RNA is between 2.0 and 2.2. Values in this range indicate that the sample is free of significant contamination by guanidine salts, phenol, carbohydrates, and other compounds that absorb at 230 nm. Values between 1.8 and 2.0 are acceptable for many applications but warrant caution for sensitive assays. Values below 1.8 indicate contamination that should be addressed before proceeding with downstream applications.

Why is my RNA 260/230 ratio low?

A low 260/230 ratio is most commonly caused by guanidine salt carryover from lysis buffers, phenol carryover from organic extraction, or carbohydrate contamination from plant or microbial sources. It can also result from measuring RNA in a buffer containing EDTA, such as TE buffer. Check your isolation protocol, ensure that washing steps were performed correctly, and verify that your blank solution matches your sample buffer.

Does a low 260/230 ratio affect downstream applications?

Yes. Contaminants that absorb at 230 nm, such as guanidine thiocyanate and phenol, can inhibit enzymatic reactions including reverse transcription, PCR, and in vitro transcription. Even if the inhibition is partial, it can introduce variability into your results and reduce the sensitivity of your assays. For RT-qPCR, low 260/230 ratios can lead to higher Ct values, reduced amplification efficiency, and increased well-to-well variability.

How can I improve the 260/230 ratio of my RNA sample?

You can improve the 260/230 ratio by performing an additional purification step. Ethanol precipitation with sodium acetate is effective for removing guanidine salts and phenol. Column-based cleanup kits are also effective and can concentrate your sample. If the ratio remains low after purification, consider re-isolating RNA from the original source material with a modified protocol.

Can I use the 260/230 ratio to assess DNA contamination?

No. The 260/230 ratio does not distinguish between RNA and DNA. DNA contamination will increase the 260 nm absorbance (and therefore the measured concentration) but will not specifically affect the 260/230 ratio. To assess DNA contamination, treat an aliquot with RNase-free DNase I and perform PCR with genomic-specific primers, or run the sample on a gel to check for high-molecular-weight DNA.

What is the difference between 260/280 and 260/230 ratios?

The 260/280 ratio compares absorbance at 260 nm to absorbance at 280 nm and primarily detects protein contamination, since proteins absorb strongly at 280 nm due to aromatic amino acids. The 260/230 ratio compares absorbance at 260 nm to absorbance at 230 nm and detects a broader range of chemical contaminants, including guanidine salts, phenol, carbohydrates, and EDTA. For pure RNA, the 260/280 ratio should be approximately 2.0-2.1, and the 260/230 ratio should be 2.0-2.2. The 260/230 ratio is generally more sensitive to buffer composition and chemical contamination than the 260/280 ratio.

Why does my 260/230 ratio vary between measurements?

Variation between measurements can result from several factors: incomplete cleaning of the pedestals between samples (carryover), air bubbles in the sample, low sample concentration (below approximately 10 ng/µL), and evaporation of the sample during measurement. To minimize variation, clean the pedestals thoroughly between measurements, pipette carefully to avoid bubbles, and ensure your sample concentration is high enough for reliable readings.

Key Takeaways

• The 260/230 ratio measures RNA chemical purity by comparing absorbance at 260 nm (nucleic acids) to absorbance at 230 nm (contaminants). Pure RNA typically gives a ratio of 2.0-2.2.
• Low 260/230 ratios are most commonly caused by guanidine salt, phenol, or carbohydrate contamination introduced during RNA isolation.
• The 260/230 ratio is more sensitive to buffer composition than the 260/280 ratio—always blank with the same buffer used to elute your RNA.
• A low 260/230 ratio does not mean your RNA is degraded; it indicates chemical contamination that may inhibit downstream enzymatic reactions.
• The 260/230 ratio cannot assess RNA integrity or DNA contamination—use gel electrophoresis, a Bioanalyzer, or DNase treatment with PCR for these purposes.
• Common measurement pitfalls include improper blanking, sample carryover on the pedestals, air bubbles, and unreliable ratios from dilute samples (<10 ng/µL).
• If the 260/230 ratio is low, purify the sample by ethanol precipitation or column cleanup, or re-isolate RNA with a modified protocol to address the specific contaminant.

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