Agarose electrophoresis of RNA requires the inclusion of denaturing agents in the gel. In the absence of denaturants, RNA assumes compact secondary structures, which distort the relationship between molecular weight and mobility. Urea, used as a denaturant in polyacrylamide gels, disrupts the hydrogen bonds which hold the agarose gel together, and alkaline conditions, used in denaturing DNA electrophoresis in agarose, hydrolyze RNA molecules. Of the variety of denaturants which can be used for RNA analysis, all are toxic or noxious to some extent. Methylmercuric hydroxide (MMH) reacts reversibly with amino groups on RNA and is a very effective denaturant. However, its toxicity and high volatility make its use inconvenient and hazardous. Aldehydes also react with RNA to disrupt base pairing and are somewhat safer than MMH. Protocols are given below for using formaldehyde or glyoxal.

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Formaldehyde Denatured RNA Gels

1. Cast Gel:
   Dissolve 1g agarose in 100ml of DI water. Heat to completely dissolve agarose crystals, and cool to 60°C.

   Add 12 ml 10X MOPS (0.2M MOPS acetate pH 7.0, 0.05M sodium acetate, 10mM EDTA) and 3.5 ml of 37% formaldehyde.

   Mix well and pour gel. Insert comb and allow to set for 30-60 minutes.
   
2. Prepare Sample:
   Mix 4.5µl of RNA (containing 10-20µg RNA), 2µl 10X MOPS, 3.5µl 37% formaldehyde and 10µl formamide.Incubate at 55°C for 15 minutes. Add 2µl of the following loading buffer, consisting of 50% glycerol, 1mM EDTA and 0.25% each bromophenol blue and xylene cyanol.
   
3. Run Gel:
   Run in 1X MOPS buffer at 10-20 V/cm for 2-3 hours, until bromophenol blue is 80-90% through the gel. Recirculate MOPS buffer to prevent pH drift. Use a peristaltic pump, or stop the run every 30 minutes and transfer buffer from cathode to anode and back.

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Formaldehyde Denatured RNA Gels

1. Cast Gel:
   Dissolve 1.2g agarose in 100ml of 10mM sodium phosphate, pH 6.9. Heat to completely dissolve agarose crystals, and cool to 60°C.

   Inhibit RNases by adding sodium iodoacetate to 10mM.

   Mix well and pour gel. Insert comb and allow to set for 30-60 minutes.
   
2. Prepare Sample:
   Dissolve 10µg of RNA in 5µl of DEPC treated water. Add 6µl 6M glyoxal, 15µl DMSO, and 3µl of 0.1M sodium phosphate, pH 6.9. Note: Glyoxal must be deionized before use. After deionization, the pH of the solution should be less than 5.

   Incubate at 50°C for 1 hour.

   Add 5µl of the following loading buffer, consisting of 50% glycerol, 10mM sodium phosphate (pH 6.9), 1mM EDTA and 0.25% each bromophenol blue and xylene cyanol.
   
3. Run Gel:
   Run in 10mM sodium phosphate, pH 6.9 at 3-5 V/cm for 3-6 hours, until bromophenol blue is 60-80% through the gel. Recirculate the buffer to prevent pH drift. Use a peristaltic pump and magnetic stir bars in the buffer chambers. If recirculation is insufficient, the pH of the buffer will rise to the point that the glyoxal will dissociate from the RNA.

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Post Electrophoretic Analysis

Stain the gel by soaking in 0.05 µg/ml ethidium bromide. Alternately, add 4 µl of 50µl/ml ethidium to the gel solution prior to pouring.

Visualize the gel on a UV light box. Two bands should appear in every lane. These are the ribosomal RNA bands. Determine their size based upon which species the RNA was isolated from, for they are useful molecular weight markers. The messenger RNA is visible as a smear from 10kb down to 1kb, and tRNA as a broad band near the end of the gel. Rinse the gel in 3 changes of deionized water to remove the Ethidium Bromide and formaldehyde prior to northern blotting.