Hi, Denaturing PAGE for miRNA detection can be quite sensitive to minor changes and from what you’ve described, two factors in your protocol likely explain why you're not seeing bands:

1. Loading Buffer with GelRed, Not Ideal for RNA

• Issue: Many GelRed-containing buffers (e.g., 6X Gel Loading Dye, Biotium) are optimized for native agarose gels, not for denaturing PAGE.
• Why it matters: GelRed is a bulky intercalating dye, and in a denaturing acrylamide system (especially with urea), it may interfere with migration and detection. It can also affect RNA integrity, especially during heating.

Fix: Use a standard denaturing RNA loading dye, typically:

• Formamide-based (e.g., 95% formamide, 0.025% xylene cyanol, 0.025% bromophenol blue, 0.5 mM EDTA)
• Heat the sample at 70–95°C for 5 minutes before loading to fully denature secondary structures.

2. Not Pre-heating the Gel (or Running it Warm)

• Why it matters: Urea gels require warmth (~50–55°C) to maintain denaturation conditions and prevent RNA secondary structures from reforming.
• Cold gels will allow miRNAs (especially GC-rich ones) to fold and migrate unpredictably or worse, stay trapped in the well.

Fix:

• Pre-warm the gel by running it for 15–30 min before loading.
• Run the gel in a warm buffer (1X TBE) at ~250–300 V ideally on a rig with active heating or in a warm room.

Additional Tips for Successful miRNA PAGE:

• Gel composition: Your 15% acrylamide, 7 M urea, 1X TBE gel is appropriate for miRNAs (~18–25 nt).
• Sample amount: Load at least 100–200 ng of total RNA for visible bands (or spike-in synthetic miRNA if probing).
• Stain after running: For better resolution and visibility, consider post-staining the gel with SYBR Gold or GelRed in TBE, instead of pre-loading dye.
• Visualize with a blue-light or UV transilluminator, depending on the stain used.

Hope this can be of help.