Does RNA Purity Affect qPCR? A260/A280 of 1.6–1.8 and Your RT-qPCR Results

RNA purity can affect qPCR, but the A260/A280 ratio alone is a poor predictor of whether it will. A sample reading 1.7 may amplify perfectly, while a sample reading 2.0 can still carry enough guanidine or ethanol to inhibit reverse transcription. The practical approach is to treat A260/A280 as a first screen, then confirm performance directly. Background on the ratio itself is in our A260/A280 guide.

What actually inhibits RT-qPCR

Guanidine salts and phenol from lysis/extraction reagents — flagged more by a low A260/A230 than by A260/A280.
Ethanol from incomplete drying of wash buffer on spin columns — not visible in absorbance ratios at all.
Sample-derived inhibitors such as heparin, heme, polysaccharides and humic substances, depending on tissue.
Genomic DNA, which does not inhibit but gives false signal unless you DNase-treat and run a no-RT control.

A simple decision path

A260/A280 ≥ 1.8 and A260/A230 ≥ ~1.8: proceed; integrity check still recommended.
A260/A280 1.6–1.8: re-measure in Tris buffer (low pH lowers the ratio). If still low, run the dilution test below before committing samples.
Low A260/A230: consider a column clean-up or an extra wash; this is the more common cause of inhibition.
Poor integrity: fix extraction and storage first — see 28S/18S interpretation.

Test for inhibition with a dilution series

Make a 5- or 10-fold dilution series of cDNA from a representative sample and amplify a reference gene. A standard-curve slope near −3.3 corresponds to about 100% efficiency; 90–110% is the usual acceptable window. If the most concentrated points give later-than-expected Cq values (the curve flattens at the top), carry-over inhibitors are present, and diluting the cDNA or cleaning the RNA will help.

Report it the MIQE way

The MIQE guidelines (Bustin et al., Clinical Chemistry, 2009) ask for RNA quantity, purity (A260/A280, A260/A230), integrity and an inhibition assessment to be reported. Keeping these four numbers for every sample makes troubleshooting far easier. For the reaction itself, a robust 2× SYBR qPCR mix and a reliable first-strand cDNA synthesis kit reduce variability between runs.

Common questions

Will an RNA A260/A280 of 1.6 or 1.7 ruin my qPCR?

Not necessarily. Modest deviations often have little effect on Cq. What matters is whether the sample contains inhibitors, which a dilution series will show.

How do I check for PCR inhibition?

Run a 5- or 10-fold dilution series of the cDNA. A slope near −3.3 (about 90–110% efficiency) with evenly spaced Cq values suggests no meaningful inhibition; Cq values that improve on dilution indicate inhibitors.

Is A260/A230 more important than A260/A280 for qPCR?

Often it is more informative, because guanidine salts, phenol and carbohydrates that inhibit enzymes show up at 230 nm. Use both, plus an integrity check.

Can I quantify cDNA with a NanoDrop?

Not reliably. After reverse transcription the reaction contains primers, dNTPs and template RNA that all absorb at 260 nm. Normalise RNA input before RT instead.

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: Bustin SA, Benes V, Garson JA, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55(4):611–622.

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