Low A260/A280 Ratio (Below 1.8) in RNA: What It Means and How to Fix It

A low A260/A280 ratio (below about 1.8) in an RNA sample means either that something is absorbing at 280 nm alongside the RNA, or that the measurement conditions are pulling the ratio down. Pure RNA typically reads around 2.0 when measured in a buffered, slightly alkaline solution. Before you re-extract, it is worth working out which of the common causes applies — several of them are measurement artefacts, not dirty RNA. For the background on why RNA and DNA have different ratios, see our complete A260/A280 guide.

The four usual causes

Acidic or unbuffered water. The ratio depends on pH. Measuring in plain water (often pH 5–6) can lower an RNA ratio by about 0.2–0.3 compared with 10 mM Tris-HCl, pH 7.5–8.5 (Wilfinger et al., BioTechniques, 1997). Same RNA, different number.
Residual phenol. Phenol absorbs strongly near 270 nm, which raises A280 relative to A260. It is common after TRIzol/TriPure-type extractions when some of the organic phase or interphase is carried over.
Protein. Protein absorbs at 280 nm, but weakly compared with nucleic acid, so it takes a lot of protein to shift the ratio. A low ratio caused purely by protein usually means a heavily contaminated prep.
Very low concentration. Below roughly 10–20 ng/µL on micro-volume spectrophotometers, small baseline errors dominate and ratios become unreliable in both directions.

How to tell which one you have

Re-blank with the same buffer the RNA is in, then re-measure the sample diluted in 10 mM Tris-HCl, pH 8.0. If the ratio comes up to ~1.9–2.1, the original reading was a pH effect.
Look at the full spectrum, not just the ratio. Clean RNA has a peak at 260 nm and a trough near 230 nm; a shoulder or shifted peak around 270 nm points to phenol.
Check A260/A230 too. Low A260/A230 together with a low A260/A280 suggests carry-over from the extraction chemistry (phenol, guanidine salts) rather than protein alone.
If concentration is low, measure a more concentrated aliquot or use a fluorescence assay for quantity, and do not over-interpret the ratio.

Fixes that work

Measure in buffer. Elute or dilute RNA in RNase-free 10 mM Tris-HCl or TE (pH 7.5–8.5) for spectrophotometry.
Column clean-up. Binding the RNA to a silica spin column, washing and re-eluting removes most phenol and salts with modest yield loss.
Extra chloroform step. For phenol-based extractions, a second chloroform extraction and careful aspiration well above the interphase reduce phenol carry-over.
Re-extract only if needed. If integrity is also poor (smeared gel, low RIN), re-extraction with fresh, properly stored tissue is a better use of time than cleaning up degraded RNA.

Will a low ratio affect my experiment?

For RT-qPCR, a ratio of 1.6–1.8 often works if the sample is not inhibitory — test this with a dilution series (see does RNA purity affect qPCR?). For RNA-seq and arrays, follow the core facility’s thresholds. Integrity matters at least as much as purity: check 28S/18S on a gel (how to read 28S/18S bands) or RIN.

Common questions

What does an A260/A280 below 1.8 mean for RNA?

It usually means something in the sample absorbs at or near 280 nm (protein or phenol), or that the RNA was measured in unbuffered, slightly acidic water, which lowers the ratio by roughly 0.2–0.3. Re-measure in a buffered solution before deciding to re-purify.

Is A260/A280 a sensitive test for protein contamination?

No. Nucleic acids absorb so strongly at 280 nm that a sample needs substantial protein before the ratio drops noticeably. A normal ratio does not prove the sample is protein-free.

Can I still use RNA with an A260/A280 of 1.6–1.7?

Often yes for RT-qPCR, if a dilution series shows no inhibition and integrity is good. For RNA-seq or microarrays, follow the core facility’s QC thresholds, which are usually stricter.

How do I fix a low A260/A280 ratio?

First re-blank and re-measure in 10 mM Tris-HCl, pH 7.5–8.5. If it is still low, clean the RNA on a silica spin column or repeat the chloroform step, taking care not to draw up the interphase.

Need cleaner RNA to start with? Changyu Biotech supplies silica-column RNA extraction kits such as the CYRN28 Plus RNA Mini Kit and a full range of RNA/DNA extraction kits for research labs worldwide. Request a quote.

Related RNA quality guides

Reference: Wilfinger WW, Mackey K, Chomczynski P. Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. BioTechniques. 1997;22(3):474–481.

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