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Low RNA yield or poor A260/230: causes and fixes

A poor A260/230 usually means salt or phenol carry-over rather than bad RNA. Low yield usually starts at lysis. Here is how to read the numbers and what to change.

Range: Nucleic acid extraction and purification3 minute readUpdated

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On this page
  1. Key points
  2. What the numbers mean
  3. Fixing a low A260/230
  4. Fixing low yield
  5. Degraded RNA
  6. Genomic DNA carry-over
  7. Questions

Key points

  • A low A260/230 usually means guanidine salts, phenol or carbohydrate carried over. The RNA itself is often fine.
  • A low A260/280 points to protein or phenol. The ratio also depends on pH, so measure in a buffered solution.
  • Low yield usually starts at lysis: incomplete homogenisation or too much starting material.
  • Absorbance cannot show integrity. Check degradation on a gel or with an RNA integrity number.

What the numbers mean

Reading RNA absorbance ratios
ReadingExpected for pure RNAIf it is low
A260/280About 2.0Protein or phenol carry-over, or an acidic blank
A260/230About 2.0–2.2Guanidine salts, phenol, carbohydrate or ethanol carry-over

Ratios also become unreliable at low concentrations, roughly below 20 ng/µL, where small absorbance errors have a large effect. Use a fluorometric assay for dilute samples.

Fixing a low A260/230

  • Silica columns. Add one more wash with the ethanol-containing wash buffer, and spin the empty column for a minute to remove every trace of ethanol before eluting.
  • Keep the membrane clean. Do not let lysate touch the column rim, where it escapes the washes.
  • Phenol-based extractions. Avoid the interphase, and wash the pellet twice in 75% ethanol.
  • Plant and polysaccharide-rich samples. Use a kit or additive designed for them. See extraction by sample type.
  • Rescue. An ethanol or lithium chloride precipitation can clean up RNA that has already been eluted.

Fixing low yield

  • Incomplete lysis. Tough tissue needs a rotor-stator or bead homogeniser. Pieces left after lysis take their RNA with them.
  • Too much starting material. Overloading a column or the lysis buffer reduces yield instead of raising it. Stay within the kit’s stated input.
  • Binding conditions. Check that ethanol was added to the wash buffer on first use and to the lysate where the protocol calls for it.
  • Elution. Apply water or buffer directly to the centre of the membrane and leave it for a minute before spinning. A second elution with the first eluate can raise the concentration.

Chemistry differs between methods: the extraction methods guide compares column, bead and phenol approaches.

Degraded RNA

Intact total RNA from mammalian cells shows sharp 28S and 18S bands at about a 2:1 ratio on a gel. Smearing toward low molecular weights means degradation. A capillary instrument gives an RNA integrity number from 1 (degraded) to 10 (intact).

  • Freeze tissue immediately in liquid nitrogen or place it in a stabilisation reagent.
  • Keep frozen samples frozen until they are in lysis buffer.
  • Use RNase-free tips and tubes, wear gloves and clean the bench with an RNase remover.

Genomic DNA carry-over

A signal in the no-RT control of a qPCR run means genomic DNA came through. Include an on-column or in-solution DNase step and design primers across an exon junction. Kits with and without DNase steps are in the extraction kit range.

Questions we get asked

Why is my A260/230 low?

Usually carry-over of guanidine salts from the lysis or binding buffer, phenol, carbohydrate or ethanol. An extra wash and a dry spin before elution often fix column preps.

Is RNA with a low A260/230 still usable?

Often yes for robust assays, but salts and phenol can inhibit reverse transcription. Test it, or clean it up by precipitation if results are poor.

What is a good RNA integrity number?

The scale runs from 1 to 10, with 10 fully intact. The value you need depends on the application; RNA sequencing is generally more demanding than a short-amplicon RT-qPCR.

Why does my A260/280 change between measurements?

The ratio depends on pH and ionic strength. Measuring in water gives lower, more variable values than measuring in a buffer at about pH 7.5.

Sources

  1. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Chomczynski P et al. Anal Biochem. 1987;162:156-9.
  2. Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Wilfinger WW et al. Biotechniques. 1997;22:474-6, 478-81.
  3. The RIN: an RNA integrity number for assigning integrity values to RNA measurements. Schroeder A et al. BMC Mol Biol. 2006;7:3.

For research use only. Protocols here are general outlines: follow your kit manual and your institution’s safety rules. See our editorial policy.

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