The A260/A280 target for miRNA and other small RNAs is the same as for total RNA — roughly 1.8 to 2.1. Absorbance at 260 and 280 nm comes from the bases, and a 22-nucleotide miRNA is made of the same bases as a 5-kb mRNA. What differs is the sample: small-RNA preparations are often low in concentration, may contain glycogen or other carriers, and are frequently mixed with larger RNA, all of which make the ratio harder to interpret. For the general rules, see our A260/A280 guide.
Why miRNA ratios are harder to trust
- Low concentration. Enriched small-RNA fractions and RNA from serum, plasma or exosomes are often below the range where micro-volume spectrophotometers give stable ratios.
- Carriers. Glycogen, linear acrylamide or carrier RNA added during precipitation contribute to the absorbance and the spectrum shape.
- Mixed populations. “miRNA” preps from total-RNA kits contain mostly rRNA and mRNA by mass; the ratio describes all of it, not the miRNA fraction.
What to measure instead
- Quantity: use a fluorescence assay designed for small RNA, or quantify by RT-qPCR against a spike-in.
- Size profile: a small-RNA electrophoresis assay shows whether the 18–25 nt fraction is present.
- Inhibition: for biofluids, add a synthetic spike-in miRNA before extraction and check its Cq across samples.
Practical tips
- Measure in buffered solution (10 mM Tris, pH ~8) and use the same buffer as the blank.
- Record whether a carrier was used, and do not compare ratios between carrier and no-carrier preps.
- If ratios are low and concentration is reasonable, follow the troubleshooting steps in low A260/A280: causes and fixes.
Common questions
What A260/A280 should miRNA have?
The same range as other RNA: about 1.8–2.1. Small RNA has no different absorbance chemistry, so the target does not change.
Is the ratio different for miRNA extraction versus total RNA extraction?
The target is the same, but small-RNA preps are often more dilute and may contain carriers, so the measured ratio is less reliable and more prone to artefacts.
Can a NanoDrop tell me how much miRNA I have?
No. Absorbance measures all nucleic acid together; it cannot separate miRNA from larger RNA or DNA. Use a small-RNA-specific assay or electrophoresis for that.
Does glycogen carrier affect A260/A280?
It can affect absorbance readings and the shape of the spectrum, especially at low RNA concentrations. Note carrier use when interpreting ratios.
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