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RNA can be completely denatured by glyoxalating the bases in the presence of DMSO, thus prohibiting base pairing. RNAs denatured in this way will migrate in agarose or PAGE gels without interference because of secondary structure. Therefore, with this system, the molecular weights of RNAs can be closely estimated by comparing them to known standards (usually glyoxalated E.coli RNA) without the use of methylmercuric hydroxide. In fact, since glyxylated DNAs and RNAs with the same molecular weights migrate the same, linear DNA, such as restriction fragments, can be used as MW standards for estimating the MWs of unknown RNAs. However, samples treated in this way should be relatively 'clean', so that the glyoxal is not depleted by contaminating junk.
Strict RNase-free technique should be used during this technique. All buffers, plasticware, and glassware should be autoclaved before use (except DMSO and glyoxal), and the electrophoresis comb and tray should be cleaned with EtOH. DON'T TOUCH THE TEETH ON THE COMB!

  1. Materials

    1. 0.1M NaH2PO4, pH 7.0 (10X GE)
      13.8 grams/liter Adjust to pH 7.0 with 50% NaOH

    2. 6M Glyoxal - Glyoxal is usually supplied at 6M (40% aqueous).
      Before use, deionize by stirring with 10g mixed resin ion exchanger (AG 501-X8) per 20ml, and store in small aliquots at -20C.

    3. Acridine orange stain
      Final Conc.
      5 mg acridine orange 10ug/ml
      0.2922 grans NaCl 10mM
      500ml

    4. 5X GE tracking dye (per 20ml)
      Final Conc.
      2ml 10X GE 10mM PO4
      10ml glycerol 50%
      0.08 grams bromophenol blue 0.4%
      8ml ddH2O

  2. Sample preparation:

    1. Mix the following in a 500ul microfuge tube:
      2.7 ul 7M glyoxal
      8.0 ul DMSO
      1.6 ul 0.2M NaH2PO4, pH 7.0
      5 ul or less RNA sample

    2. Incubate capped at 56C for 1 hr. While the samples are incubating, prepare the gel & apparatus as described below.

  3. Gel:

    1. Use a 1.0-2.0% agarose gel (or 5-12% PAGE) made in GE. The gel should be poured while hot - about 85C.
    2. Recirculate the buffer by running the gel with the buffer trays on top of stir-motors, with gentle stirring, and with a peristaltic pump exchanging buffer between the trays.
    3. Electrophorese at 1-5 V/cm until the dye reaches about 3/4 of the way down the gel.
    4. If the gel isn't going to be transferred to nitrocellulose, stain the gel for 1 hr in acridine orange, then destain by soaking in ddH2O for 1-1.5 hr. Examine under a UV light. To photograph, use Kodak Wratten filters; #12 for both green and red bands, #40 for green bands (native DNA, rRNA),and #29 for red bands (denatured RNA & DNA, non-rRNA).

  4. Northern Blotting glyoxylated RNA or DNA

    RNA normally has a low affinity for nitrocellulose since native RNA has extensive secondary structuring. Thus, older methods were developed for Northern blotting using DBM-activated paper to bind native RNA. However, glyoxylated nucleis acids are thoroughly denatured and will readily bind nitrocellulose. Therefore both RNA and DNA can be easily transferred from agarose gels to nitrocellulose, after glyoxylation.

    1. Wet a pre-cut sheet of nitrocellulose in ddH2O, then soak in 20X SSC for at least 5 min.
    2. Lay a wetted (in 20X SSC) 3MM paper wick over an upside-down gel tray in a dish of 20X SSC. Ovr this lay a piece of 3MM paper (again, pre-wetted in 20X SSC) cut exactly the same size as the gel. Roll a pipette over the paper to remove ALL of the air bubbles.
    3. Place the gel directly after electrophoresis (i.e. untreated & unstained) face-down onto the wet 3MM paper. Again, roll out any trapped air bubbles with a pipette.
    4. Place the nitrocellulose filter on top of the gel, being careful not to trap any air under the nitrocellulose (or roll them out as before).
    5. Cover the filter with 2 layers of pre-wetted (20X SSC) 3MM paper cut the same size as the gel. For the last time, remove trapped air by rolling over the paper with a pipette.
    6. Surround the gel on all four sides with strips or saran-wrap, so that the dish and wick are covered. The sara-wrap should butt up against the gel on all four sides.
    7. Lay a thick stack of paper towels onto the top 3MM paper sheet. Over this, lay a glass plate large enough to cover the area of the gel. On top, place a 500ml flask filled with water, as a weight.
    8. Replace the paper towels as they become wet, whenever convenient. The transfer is complete after 16-24 hrs.
    9. Remove the nitrocellulose filter & allow it to air-dry. DO NOT WASH BEFORE BAKING! Bake at 80C in a vacuum oven for 2hr.
    10. Bring 500ml 10mM Tris, pH8, to a boil over a bunsen burner, then pour this hot solution into a plastic tray. Drop the filter into the hot Tris, submerge the filter, then wait for the solution to cool. Remove the filter, air dry, and store wrapped in foil at RT until use. This step reverses any residual glyoxylation, allowing more efficient hybridization.

  5. Staining Northerns with Methylene blue

    After autoradiography, Northerns may be stained with methylene blue to visualize the transferred RNA. This is especially useful, since glyoxylated RNAs should not be stained before transfer to nitrocellulose from agarose gels.

    1. Soak the nitrocellulose filter in 5% acetic acid for 15 min.
    2. Transfer the filter to methylene blue stain (0.5M Na acetate, pH 5.2 + 0.04% methylene blue), and soak for 10 min.
    3. Rinse the filter for at least 10 min. in ddH2O.

McMaster and Carmichael 1977 PNAS USA 74:4835
Maniatis, et al 1982 "Molecular Cloning" CSH
Davis, et al 1980 "Advanced Bacterial Genetics" CSH pp156-158
Thomas 1980 PNAS USA 77(9):5201

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