You can check total RNA integrity on an ordinary, non-denaturing agarose gel if you denature the sample before loading and keep the run short and clean. The goal of this quick gel is a go/no-go answer — are the 28S and 18S rRNA bands sharp, and is there a smear? — not accurate sizing. For interpreting the result, see 28S:18S rRNA ratio and what degraded RNA looks like.
Quick protocol (native agarose, total RNA)
- Gel: 1–1.2% agarose in fresh 1× TAE or TBE, about 3–5 mm thick, with stain in the gel or post-stain.
- Equipment: a clean tank, tray and comb reserved for RNA if possible; rinse with fresh buffer before use.
- Sample: 0.5–1 µg total RNA mixed with a formamide-containing RNA loading dye; heat at 65–70 °C for 5–10 min, then chill on ice and load immediately.
- Run: about 5–7 V/cm for 20–40 min — long enough to separate 28S from 18S, short enough to limit RNase exposure.
- Image: look for two sharp rRNA bands with 28S brighter than 18S and a faint low-molecular-weight band (5.8S/5S/tRNA).
Why heat-denature even on a native gel
RNA folds into secondary structures that change how fast it migrates. Heating in formamide unfolds it so bands are sharper and more reproducible. Without this step you may see extra or fuzzy bands that are conformers of the same RNA, not degradation.
RNase control without overdoing it
- Wear gloves, use filter tips and keep samples on ice until loading.
- Use freshly prepared buffer; old buffer that has sat in a shared tank is the most common RNase source.
- Load promptly after heating and run immediately.
- For precious samples or long runs, decontaminate equipment with an RNase-removal solution and use RNase-free water for buffers.
When you need a denaturing gel instead
- Accurate size of a transcript or IVT product — use a formaldehyde/MOPS agarose gel with an RNA ladder.
- Northern blotting — denaturing agarose is standard.
- Small RNAs or short IVT RNA — use denaturing urea-polyacrylamide gels.
- Quantitative integrity (RIN/RQN) — use capillary electrophoresis. See what a slab gel can and cannot tell you.
Common questions
Can you run RNA on a normal (non-denaturing) agarose gel?
Yes, for a quick integrity check. Heat the RNA in formamide-containing loading dye before loading and run a short, clean gel. For accurate sizing or Northern blots, use a denaturing formaldehyde gel.
What agarose percentage is best for total RNA?
About 1–1.5% resolves the 28S and 18S rRNA bands well for a quick check.
Do I need RNase-free water and buffer?
Use fresh running buffer and a clean tank and comb. For a short quality-check run, freshly made buffer in clean equipment is usually adequate; for long runs or precious samples, use RNase-free reagents and decontaminated equipment.
Should mRNA be run on agarose or polyacrylamide?
Long mRNAs (hundreds of nucleotides to several kb) are run on agarose. Short RNAs and small IVT products (below a few hundred nucleotides) resolve better on denaturing urea-polyacrylamide gels.
How thick should the gel be?
Thinner gels (about 3–5 mm) give sharper bands and need less stain; thick gels blur bands and raise background.
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