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Short answer
RNA is run in agarose gels for one main reason: a fast look at how much RNA there is and whether it is intact. Two formats are in common use. A nondenaturing agarose gel in TBE is a routine quantity-and-integrity check, but it should not be used to size RNA accurately. A denaturing formaldehyde-MOPS gel is the format Thermo Fisher describes for the same check and for samples headed to a Northern blot. Intact eukaryotic total RNA shows sharp 28S and 18S rRNA bands, with the 28S band about twice as intense as the 18S.
Definition and scope
Agarose gel electrophoresis of RNA separates RNA molecules by size in an electric field. RNA is negatively charged, so it migrates toward the anode, and the gel matrix slows larger molecules more than smaller ones (Rio et al., 2010). This page covers choosing between the two agarose formats and reading the finished lane. It does not repeat DNA-focused gel basics, which are covered in agarose gel electrophoresis basics, or the general principle, covered in what is gel electrophoresis.
Agarose is not the only gel option for RNA. Rio et al. (2010) note that denaturing acrylamide gels suit RNAs up to roughly 600 nucleotides, while agarose suits longer RNAs such as the mRNAs analysed by Northern blotting.
How RNA is run in practice
Thermo Fisher’s protocol page for agarose gel electrophoresis of RNA gives the denaturing format in these terms: a 1% agarose gel made with 1X MOPS running buffer and 37% formaldehyde (12.3 M), covered with 1X MOPS buffer once set. The 10X MOPS stock is 0.4 M MOPS at pH 7.0, 0.1 M sodium acetate and 0.01 M EDTA. Samples of 1 to 3 µg RNA are mixed with formaldehyde load dye and heated at 65 to 70 °C, and the gel is run at 5 to 6 V/cm until the bromophenol blue has moved at least 2 to 3 cm or about two-thirds of the gel length.
The nondenaturing alternative is the routine TBE agarose protocol published in Cold Spring Harbor Protocols (Rio et al., 2010), described there as a way to check RNA quantity and integrity before downstream use.
Formaldehyde is a hazard in this format. Thermo Fisher states that it is toxic through skin contact and inhalation of vapors, and the gel work belongs in a chemical fume hood. For exposure limits and monitoring rules, see the OSHA formaldehyde standard (29 CFR 1910.1048) and your institution’s chemical hygiene plan.
Denaturing versus nondenaturing: a sourced comparison
| Question | Nondenaturing TBE agarose gel | Denaturing formaldehyde-MOPS agarose gel |
|---|---|---|
| What the source says it is for | Checking RNA quantity and integrity before downstream use (Rio et al., 2010) | Checking RNA, including samples prepared for Northern analysis (Thermo Fisher) |
| Accurate sizing | Not recommended: the procedure “should not be used to determine size with accuracy” because RNA does not stay fully extended (Rio et al., 2010) | Described by Thermo Fisher as the format for samples prepared for Northern analysis (see the Northern blot guide below) |
| Sample preparation | TBE agarose gel (Rio et al., 2010) | Formaldehyde load dye at 0.5X for a quick check or 3X for full denaturation such as a Northern; heat 65 to 70 °C for 5 minutes (simple check) or 15 minutes (Northern) (Thermo Fisher) |
| Chemical hazard handling | Uses TBE buffer rather than formaldehyde (Rio et al., 2010) | Formaldehyde in the gel: fume hood required (Thermo Fisher) |
The table is the practical gain of this page: the format question is answered by what the gel is for. Both sources describe a quantity-and-integrity check. If size information matters, the nondenaturing protocol itself warns against accurate sizing; the denaturing format adds a formaldehyde hazard to manage.
Reading the lane: a checklist
Thermo Fisher describes four patterns for total or enriched RNA stained on a denaturing gel.
- Intact eukaryotic total RNA: sharp 28S and 18S bands, with the 28S band approximately twice as intense as the 18S band.
- Partially degraded: a smear, no sharp rRNA bands, or sharp bands that lack the 2:1 intensity pattern.
- Completely degraded: a very low molecular weight smear.
- Poly(A)-selected RNA: no strong rRNA bands and a smear from about 6 kb down to 0.5 kb. A missing 28S and 18S pair is expected here and is not a sign of failure.
Two checks come before interpretation. First, loaded mass: Thermo Fisher states that at least 200 ng of RNA must be loaded to see it with ethidium bromide, whereas SYBR Gold detects about 1 ng and SYBR Green II about 2 ng. An empty-looking lane may only mean too little RNA for the stain. Second, stain consistency: if ethidium bromide is added in the load dye (10 µg/ml), the same amount must go into every sample, including the size marker, because it affects RNA migration.
Where it matters for labs
A gel is a coarse quality gate. The 28S to 18S intensity ratio seen on a gel is not the same thing as an RNA Integrity Number; for the instrument-based score and how it is read, see RNA integrity number (RIN). For the preceding step, RNA extraction basics covers what comes before the gel check. When the sample is going to a Northern blot, the denaturing format and the heavier 3X dye and 15-minute heating step are the ones Thermo Fisher ties to that use; see Northern blot.
Common misunderstandings
- “A band at the right place proves the RNA size.” On a nondenaturing gel it does not; the protocol itself advises against accurate sizing.
- “No 28S and 18S bands means the prep failed.” Not for poly(A)-selected RNA, which shows no strong rRNA bands by design.
- “Any agarose gel will do for any RNA.” Short RNAs of about 600 nucleotides or less are the territory of denaturing acrylamide gels (Rio et al., 2010).
- “The marker can skip the stain.” If ethidium bromide goes in the samples, the marker needs the same amount.
FAQ
Can I size RNA accurately on a nondenaturing agarose gel?
No. The published TBE protocol states it should not be used to determine size with accuracy, because RNA does not stay fully extended (Rio et al., 2010).
What should intact total RNA look like?
Two sharp bands, 28S and 18S, the 28S about twice as intense as the 18S (Thermo Fisher).
How much RNA do I need to see on a gel?
At least 200 ng for ethidium bromide staining; about 1 ng with SYBR Gold and about 2 ng with SYBR Green II (Thermo Fisher).
Do I need a fume hood for a denaturing RNA gel?
Thermo Fisher states that formaldehyde is toxic through skin contact and inhalation of vapors and that the work should be done in a chemical fume hood (Thermo Fisher).
Sources
Date checked: 2026-10-29.
- Thermo Fisher Scientific, “Agarose Gel Electrophoresis of RNA” protocol page: https://www.thermofisher.com/hk/en/home/references/protocols/nucleic-acid-purification-and-analysis/rna-protocol/agarose-gel-electrophoresis-of-rna.html
- Rio DC, Ares M Jr, Hannon GJ, Nilsen TW. “Nondenaturing agarose gel electrophoresis of RNA.” Cold Spring Harbor Protocols 2010(6):pdb.prot5445. doi:10.1101/pdb.prot5445: https://pubmed.ncbi.nlm.nih.gov/20516183/
- OSHA, Formaldehyde, 29 CFR 1910.1048: https://www.osha.gov/laws-regs/regulations/standardnumber/1910/1910.1048
