Most strange A260/A280 readings on micro-volume spectrophotometers such as the NanoDrop come from the measurement, not the RNA. Negative ratios, values that change on every repeat, or ratios far outside 1.6–2.3 usually trace back to the blank, the pedestal, bubbles or a sample that is too dilute. Work through the checklist below before you decide the RNA itself is bad. What the ratio means is covered in our A260/A280 guide.
Checklist: fix the measurement first
- Blank with the sample’s own buffer. Blanking with water and measuring RNA in elution buffer (or vice versa) shifts absorbance, especially at 230 and 280 nm.
- Clean both pedestals with water and a lint-free wipe before blanking and between samples; dried residue gives false absorbance.
- Mix and spin the sample so the drop is homogeneous and free of bubbles; load 1–2 µL and make sure the column forms properly.
- Re-blank periodically during long sessions, and re-measure any outlier.
- Mind the concentration. Below ~10–20 ng/µL, ratios are dominated by noise. Above the instrument’s linear range, dilute and re-measure.
Read the spectrum, not just the numbers
- Clean RNA: smooth curve, peak at 260 nm, trough around 230 nm, near-zero absorbance at 320–340 nm.
- Peak shifted toward 270 nm: phenol carry-over.
- High absorbance at 230 nm: guanidine salts, EDTA or carbohydrate carry-over (low A260/A230).
- Raised baseline at 320–340 nm: particles, turbidity or a dirty pedestal.
If the measurement is sound and the ratio is still off
Then the sample really does carry contaminants. Low ratios are covered in low A260/A280: causes and fixes; high readings in is a high ratio good or bad?. Remember that absorbance cannot detect degradation or gDNA — check integrity on a gel (what degraded RNA looks like).
Common questions
Why is my A260/A280 negative or nonsensical?
Almost always a blank problem: the blank was in a different buffer, the pedestal was dirty when blanking, or the sample is so dilute that A280 is near zero. Clean, re-blank with the sample’s own buffer, and re-measure.
Why do repeat readings of the same sample differ?
Bubbles, uneven sample columns, evaporation and very low concentration all cause scatter. Mix gently, spin down, load fresh 1–2 µL drops and take at least two readings.
What concentration is too low for a reliable ratio?
On micro-volume instruments, ratios become noisy below roughly 10–20 ng/µL of RNA. Quantity may still be usable, but treat the ratio with caution.
Should I use baseline correction at 320/340 nm?
It helps correct for turbidity and light scatter, but a large signal at 320–340 nm is itself a warning that the sample contains particles or the pedestal is dirty.
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