Hi! We are trying to set up an EMSA assay with RNA and an RNA-binding protein. Initially we tested various RNA concentrations without protein to determine what concentration to use moving forward, but none have shown up. We use 0.5 x TBE and a 4% agarose gel, though we have also tried a few other buffers and gel percentages. We've tried making the gel with ethidium bromide, as well as post-staining with ethidium bromide or SYBR gold. Our RNA sequences are short (10-15 oligonucleotides) but we have tested concentrations far above the detection limit (up to ~40 ng). The DNA ladder we use appears normally. Does anyone have any ideas on why these might not show up?
It sounds like you're troubleshooting RNA visualization in agarose gels before proceeding with EMSA, and you're doing a lot of things right. Since your RNA is short (10–15 nt), there are a few key reasons why it might not be visible: 1. Gel Type and Resolution Agarose is not ideal for very short oligonucleotides (under ~50 nt). Short RNA (10–15 nt) migrates very quickly and may run off the gel or not resolve well. Recommendation: Use urea-denaturing polyacrylamide gel electrophoresis (PAGE) instead (e.g., 15–20% acrylamide with 7 M urea). This will give much better resolution and retention of short RNA. 2. Staining Sensitivity Ethidium bromide is not very sensitive for short single-stranded RNA. SYBR Gold is better, but short oligos still bind dyes less efficiently than longer RNAs or DNA.
Recommendation: Stick with SYBR Gold or even try radiolabeling or fluorescent labeling if possible, especially for EMSA. 3. RNA Degradation Although unlikely if your ladder looks fine, short RNA can degrade easily and smear or disappear. Recommendation: Ensure RNase-free conditions, use fresh aliquots, and consider running a positive control (known good RNA). 4. Sample Loading and Migration Short oligos may run off even a 4% agarose gel, especially in long runs or with high voltage. Recommendation: Try a very short run time, or better yet, switch to PAGE for these small sizes. 5. RNA Structure RNA might be forming secondary structures that affect staining or migration. Recommendation: Heat-denature before loading (e.g., 95°C for 3–5 min in formamide or loading dye), then snap cool on ice. Summary: Use denaturing PAGE (15–20% acrylamide + urea). Use SYBR Gold or consider 5' fluorescent/radioactive labeling. Ensure RNase-free handling and proper denaturation. Avoid agarose for
Try 1% agarose gel. 100mV for 10- 20 min. Use atleast 200ng to load. And use autoclaved MilliQ to prepare buffer and gel. Let me know if this helps.
To the best of my knowledge you can not see RNA directly on an agarose gel, especially mRNA. However, if the subject to visualize are structural RNA, the EtBr may intercalate between the helices grooves and help in visualization. You are able to see the ladder normally, because it is probably a standard DNA ladder. When checking for total RNA on an agarose gel, a 2--3% gel in 1X TAE should show 3 distinct bands of ribosomal RNA. Also, check for the quality of RNA extracted(260/280 ratio), which is ideally >1.9 for RNA.
Maybe this helps.
Thank you
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