If an RNA sample reads A260/A280 ≈ 1.8 against a pH-buffered blank, many protocols still stamp it “pure.” Under those solvent conditions that number sits on the DNA benchmark (~1.8), not on fully deprotonated RNA (~2.1). Residual protein, phenol, genomic DNA, and an acidic water blank all pull the same ratio down. This page is a laboratory decision guide: how to read A260/A280 with A260/A230, what the ratio cannot tell you (integrity), and when 1.8 means change the blank or re-extract—not load the sample into RT-qPCR.
This page is for molecular biology labs that already have a UV reading and need a next action. It is not a kit shopping list and not a diagnostic protocol. All guidance below is for research-use nucleic acid quality control.
Updated 28 August 2026.
What A260/A280 actually measures
Ultraviolet absorbance of a nucleic acid solution follows the Beer–Lambert relation:
Aλ = log10(I0 / I) = ελ · c · ℓ
where Aλ is absorbance at wavelength λ, I0 and I are incident and transmitted intensity, ελ is the wavelength-specific extinction coefficient, c is concentration, and ℓ is pathlength in cm.
Purine and pyrimidine bases have conjugated rings. Their π→π* transitions produce a strong peak near 260 nm. In a 1 cm cuvette, A260 = 1.0 is the usual working equivalent of about 50 µg/mL dsDNA, 40 µg/mL RNA, or 33 µg/mL single-stranded oligonucleotide. Proteins peak near 280 nm because of tryptophan and tyrosine (with smaller input from phenylalanine and cystine). The ratio A260/A280 exists because those two peaks do not coincide.
The ratio did not start as an RNA QC metric. Warburg and Christian used A280/A260 in 1941 to correct nucleic acid contamination in protein (enolase from yeast). Molecular biology later inverted the same two wavelengths to watch protein contamination in nucleic acid. That history matters: the assay is a two-component compromise, not a complete purity certificate.
On a NanoDrop-class pedestal the physical pathlength is usually 0.1 cm or 0.05 cm. The ng/µL the software prints is already scaled to a 1 cm equivalent. The A260/A280 ratio is unitless and still needs the same blank as the eluate. Do not treat a water-blanked 1.8 on a pedestal and a Tris-blanked 2.1 in a cuvette as two different RNA chemistries.
Why pure RNA does not share DNA’s 1.8
In neutral-to-slightly alkaline buffer, highly purified dsDNA is typically near A260/A280 ≈ 1.8. Highly purified single-stranded RNA is higher, commonly ~2.0–2.2 when the bases are deprotonated.
Several structural facts drive the gap; none of them is a kit slogan:
- Uracil versus thymine. RNA uses uracil; DNA uses 5-methyluracil. The missing C-5 methyl group changes the pyrimidine absorption envelope. Uracil contributes relatively more at 260 nm than thymine does.
- The 2′-OH. Ribose carries a hydroxyl at carbon 2′; deoxyribose does not. That local polarity shifts the nucleobase spectrum slightly.
- Hypochromicity. B-form DNA stacks bases tightly and suppresses UV absorbance. Total RNA is a mix of stacked and unstacked regions, so more 260 nm signal survives per mole of base.
- Base composition. Total RNA is mostly rRNA, often GC-richer than average genomic DNA. G and C have higher intrinsic 260/280 ratios than A and T.
A globular protein such as BSA has a mass extinction at 280 nm on the order of 0.6–1.0 mL·mg⁻¹·cm⁻¹. RNA at 260 nm is roughly 20–40 times stronger per unit mass. That is why stripping protein makes A260/A280 rise—and why a “normal” ratio is a weak protein detector (Glasel, 1995; Manchester, 1995).
The solvent is part of the measurement
Wilfinger, Mackey, and Chomczynski (1997) showed that the same RNA yields different A260/A280 values when the diluent changes. Unbuffered water (including many DEPC-treated stocks) absorbs atmospheric CO2, forms carbonic acid, and drifts toward pH ~5–6.
At acidic pH, ring nitrogens on A, C, and G protonate. Absorbance at 260 nm falls and absorbance at 280 nm rises, so the ratio drops. Pure RNA measured in acidic water can look like “protein-contaminated RNA” when the only problem is the blank.
Practical rule:
- Blank and dilute in the same buffer the RNA was eluted in.
- Prefer 10 mM Tris-HCl, pH 7.5 or TE (10 mM Tris, 1 mM EDTA, pH 8.0).
- Do not blank with water and then measure a TE eluate.
- If the reading is 1.5–1.7 and the RNA is in water, re-measure in Tris/TE before you throw the prep away.
Directionally: acidic unbuffered water depresses the ratio; Tris/TE near pH 8 restores it toward the RNA benchmark. Exact cells in a vendor table are not a substitute for matching your own blank.
Why 1.8–2.0 became “good enough” RNA
A buffered RNA reading of 1.8 is not the theoretical RNA spectrum. It usually means one or more of:
- Genomic DNA co-purified. DNA’s baseline is ~1.8. A DNA-heavy mix lands near 1.8–1.9 even when protein is low.
- Protein or phenol carryover. Phenol absorbs near 270 nm, inflates A260 (so yield is overestimated), and depresses both 260/280 and 260/230.
- The blank is still acidic water. The sample may be fine; the assay is not.
The 1.8–2.0 window survived for three operational reasons, not because biophysics changed.
Phenol–chloroform workflows. The acid guanidinium–phenol–chloroform method (Chomczynski and Sacchi, 1987), commercialized as TRIzol-type reagents, is fast and harsh. Without aggressive cleanup it leaves interphase protein and phenol. Labs that needed usable RNA from that chemistry accepted 1.8–2.0 rather than discard every tube.
Spin-column specifications. Early silica kits did not all clear gDNA and protein to the same degree. Printing “1.8–2.1 acceptable” on a protocol reduces arguments with users. It does not rewrite the RNA absorption spectrum.
Water blanks. Measuring in DEPC water made even clean RNA look like 1.6–1.9, which trained a generation of protocols to treat those numbers as normal (Wilfinger et al., 1997).
A useful working band in matched Tris/TE, pH ~8, not in water:
| Buffered A260/A280 | What to do next |
|---|---|
| < 1.7 | Do not treat as “pure RNA.” Check blank pH, then protein/phenol/gDNA. Re-extract or clean up. |
| 1.7–1.9 | DNA-like or still dirty. Run A260/A230, consider DNase or a gDNA-removal step, do not skip a second method if the assay is RT-qPCR. |
| 2.0–2.2 | Chemically plausible for RNA in this solvent. Still not a RIN. Still not “no protein.” |
| > 2.3 on a microvolume pedestal | Usually noise, low concentration, or an overly alkaline blank—not proof of degradation. |
These bands are decision aids. They are not a certificate of composition.
A260/A280 is blind to several contaminants that kill enzymes
Because nucleic acids out-absorb proteins, Glasel (1995) and Manchester (1995) noted that protein must be a large mass fraction before 260/280 falls. A sample can read “fine” and still hold enough protein or salt to inhibit reverse transcriptase.
That is why A260/A230 belongs on the same printout. Pure nucleic acids typically sit near 2.0–2.2 at 260/230. Many extraction chemicals peak near 230 nm.
| Contaminant | Where it eats the spectrum | What you see | Downstream risk |
|---|---|---|---|
| Protein | 280 nm | 260/280 falls only when protein is already substantial | Polymerase / RNase issues |
| Guanidinium (GITC, guanidine HCl) | ~230 nm | 260/230 collapses; 260/280 may look normal | RT and PCR inhibition |
| Phenol | ~270 nm | Both ratios fall; A260 overestimates RNA | Enzyme denaturation, wild Ct |
| Polysaccharides / glycogen | ~230 nm | 260/230 falls | Poor solubilization, inhibition |
| Dust / bubbles / lipids | scatter, including 320 nm | Unstable ratios | Baseline lies |
| Acidic water blank | protonation, not a chemical spike | Fake-low 260/280 | Unnecessary re-extraction |
Huberman (1995) argued for watching the short-UV side of the spectrum, not only 260 and 280. Subtracting A320 (scatter) before forming ratios is still the cheap way to stop a dirty pedestal from inventing yield.
A260 itself is also not a structure assay. Breaking the phosphodiester backbone does not destroy the bases, so degraded RNA still reads at 260 nm. A beautiful 2.1 ratio can be a soup of oligomers. Integrity is a RIN / RQI / electropherogram problem (Schroeder et al., 2006; Fleige and Pfaffl, 2006). MIQE treats spectrophotometric purity and integrity as separate checkpoints (Bustin et al., 2009). A ratio above 2.3 on a NanoDrop-class instrument, especially below ~20 ng/µL, is almost always baseline noise, not “extra-pure” or “extra-degraded.”
When the methods gap is phenol and gDNA — one plant kit
The 1.8-as-pure habit is not a branding argument. It is a methods-stage mismatch.
Acid guanidinium–phenol lysis is the right tool when the sample needs aggressive disruption. It is the wrong tool to pretend that a 1.8 ratio in TE means RNA-only. Phenol and interphase protein sit in the same UV windows you are using for QC.
Silica spin columns with a dedicated genomic-DNA elimination column close a different gap: they skip phenol/chloroform, they bind gDNA on a first membrane, and they leave RNA for a second silica membrane. That architecture is for the research question “is this isolate RNA-biased enough to take into RT-qPCR,” not for clinical diagnosis.
Changyu Bio publishes one typical UV window in a manual: the Plus Complex Plant RNA Mini Kit (Cat# CYRN53). After the gDNA-elimination column and phenol-free washes, the manual states a typical OD260/OD280 of 2.0–2.2. That is a typical range on that plant protocol (including difficult tissues such as Dendrobium), not a lot certificate, not a promise for every operator, and not a specification copied onto other kits. The same manual warns that trace gDNA can remain for highly sensitive assays; intron-spanning primers or an extra DNase step are still the honest next move. Store and ship those buffers at room temperature—the manual says 4 °C or −20 °C precipitates them.
Use that plant kit when the sample is plant and the QC question is chemical purity plus gDNA reduction. Do not read 2.0–2.2 as a company-wide RNA slogan. Other extraction routes (phenol lysis plus silica cleanup, or mammalian spin columns) have to be judged on their manuals and on the same buffered UV + RIN logic above. See the RNA and DNA extraction kit list for the rest of that family.
What to do at the bench this week
- Match the blank. Elution buffer in the reference cell; same pH, same EDTA.
- Stay in the linear window. On a 1 cm cuvette, trust A260 roughly 0.1–1.0. Below that, use a fluorometric dye (RNA-selective) instead of inventing a ratio from noise. Above that, dilute. On a pedestal, treat readings below ~20 ng/µL as instrument floor.
- Record both ratios plus A320. If 260/230 is < 1.8, look at guanidinium and phenol before you celebrate 260/280.
- Do not use 260 nm as RIN. If the experiment is RT-qPCR, RNA-seq, or any length-sensitive assay, add capillary electrophoresis. MIQE asked for this in 2009; it is still the split that matters.
- Read 1.8 in TE as a flag, not a pass. Re-blank in Tris/TE if the first shot was water. If it stays 1.8, assume DNA or leftover organics until a second method says otherwise.
- Low concentration lies. Pedestal ratios on < 20 ng/µL RNA are often instrument floor, not sample chemistry.
Common questions
Is an RNA A260/A280 of 1.8 a purity pass? In matched Tris/TE near pH 8, 1.8 is the DNA-like band. Re-blank if the first shot was water. If it stays 1.8, assume genomic DNA or leftover organics until a second method says otherwise.
Why is RNA 260/280 about 2.0, not 1.8? Deprotonated RNA in alkaline buffer typically sits near 2.0–2.2 because of uracil versus thymine, the 2′-OH, weaker hypochromicity than B-form DNA, and rRNA base composition. A 1.8 RNA reading is usually solvent, DNA, or carryover—not “RNA being DNA-like by nature.”
Can A260/A280 replace RIN? No. Broken RNA still absorbs at 260 nm. Integrity is a RIN / RQI / electropherogram measurement. MIQE treats spectrophotometric purity and integrity as separate checkpoints.
Bottom line
Pure RNA in Tris/TE near pH 8 does not live at 1.8. That number is the DNA-like, phenol-tolerant, water-blanked compromise that extraction history trained people to accept. A buffered 2.0–2.2 with a healthy 260/230 means the chemistry is in a plausible RNA band. It still does not mean intact transcripts, and it still does not mean zero protein. Pair pH-controlled UV with a fluorometric count and a RIN if the downstream assay cares.
Research use only. Not for diagnostic, therapeutic, or clinical use.
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References
- Wilfinger, W. W., Mackey, K., & Chomczynski, P. (1997). Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. BioTechniques, 22(3), 474–481. https://doi.org/10.2144/97223st01
- Warburg, O., & Christian, W. (1941). Isolierung und Kristallisation des Gärungsferments Enolase. Naturwissenschaften, 29(39), 589–590. https://doi.org/10.1007/bf01482279
- Chomczynski, P., & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry, 162(1), 156–159. https://doi.org/10.1016/0003-2697(87)90021-2
- Glasel, J. A. (1995). Validity of nucleic acid purities monitored by 260nm/280nm absorbance ratios. BioTechniques, 18(1), 62–63. PMID: 7702855
- Manchester, K. L. (1995). Value of A260/A280 ratios for measurement of purity of nucleic acids. BioTechniques, 19(2), 208–210. PMID: 8527139
- Huberman, J. A. (1995). Importance of measuring nucleic acid absorbance at 240 nm as well as at 260 and 280 nm. BioTechniques, 18(4), 636. PMID: 7598897
- Fleige, S., & Pfaffl, M. W. (2006). RNA integrity and the effect on the real-time qRT-PCR performance. Molecular Aspects of Medicine, 27(2–3), 126–139. https://doi.org/10.1016/j.mam.2005.12.003
- Schroeder, A., Mueller, O., Stocker, S., Salowsky, R., Leiber, M., Gassmann, M., Lightfoot, S., Menzel, W., Granzow, M., & Ragg, T. (2006). The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Molecular Biology, 7, 3. https://doi.org/10.1186/1471-2199-7-3
- Bustin, S. A., Benes, V., Garson, J. A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M. W., Shipley, G. L., Vandesompele, J., & Wittwer, C. T. (2009). The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry, 55(4), 611–622. https://doi.org/10.1373/clinchem.2008.112797
- Lucena-Aguilar, G., Sánchez-López, A. M., Barberán-Aceituno, C., Carrillo-Ávila, J. A., López-Guerrero, J. A., & Aguilar-Quesada, R. (2016). DNA source selection for downstream applications based on DNA quality indicators analysis. Biopreservation and Biobanking, 14(4), 264–270. https://doi.org/10.1089/bio.2015.0064