Fong:RNA gel electrophoresis/Denaturing

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Gel electrophoresis separates RNA in essentially the same way as DNA, but since RNA often folds into a native conformation, it is necessary to denature the RNA strands if separation by size is needed. The RNA is denatured in this protocol by incubating with fomaldehyde at 60°C.

As with all RNA work, great care must be taken to avoid any contamination with RNAse.


  • Agarose
  • DEPC
  • 10x MOPS buffer, made using DEPC-treated water (see below)
  • Formaldehyde
  • Formamide
  • RNAse Away spray
  • RNAse free filter pipette tips
  • Gel casting tray and comb
  • Gel box
  • Electrophoresis power supply


Treating water with DEPC to remove RNAse

  1. For 1% DEPC solution, add 2ml DEPC to 2L bottle and fill with nanopure H2O.
  2. Shake well and allow solution to incubate for at least 4 hours, or overnight if possible.
  3. Autoclave bottles on liquid cycle or for 10min at 120°C to remove traces of DEPC.
  4. Spray all glassware, casting tray, and gel box with RNAse Away and rinse well with DEPC treated water.

Denature RNA in buffer

  1. Combine:
    • 5μL RNA sample (up to 30μg)
    • 1μL 10x MOPS
    • 3.5μL formaldehyde
    • 10μL formamide
  2. Incubate at 60°C for 15-20min

Make Denaturing Gel (medium, 50mL gel)

  1. In a DEPC-H2O rinsed flask, combine
    • 0.5g Agarose
    • 4.2mL 10x MOPS
    • 37.5mL DEPC-H2O
    • 3.7μL formaldehyde
  2. Microwave for ~1.5 min until melted, cool to ~60°C
  3. Add 3.5μL ethidium bromide, and pour into casting tray.

Loading and running gel

5μL of the orange formaldehyde loading dye works for 1-30μg RNA. Modify amounts of RNA sample and dye to get strong enough bands, depending on the sample type and experiment requirements. Run the gel at 120V for 45-50min, or, for better resolution, 100V for 1hr.


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