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        Agarose Gel Electrophoresis of RNA

        互联网

        1040

        实验试剂

         

        Gel Loading Buffer II

        Gel Loading Solution

        NorthernMax Formaldehyde Load Dye

        NorthernMax-Gly Sample Loading Dye

        实验步骤

         

        1. Prepare the gel.

        1) Heat 1 g agarose in 72 ml water until dissolved, then cool to 60°C.

        2) Add 10 ml 10X MOPS running buffer, and 18 ml 37% formaldehyde (12.3 M).

        3) Pour the gel using a comb that will form wells large enough to accommodate at least 25 µl.

        4) Assemble the gel in the tank, and add enough 1X MOPS running buffer to cover the gel by a few millimeters. Then remove the comb.

        2. Prepare the RNA sample.

        1) To 1-3 µg RNA, add 0.5-3X volumes Formaldehyde Load Dye.

                   i. To simply check the RNA on a denaturing gel, as little as 0.5X Formaldehyde Load Dye can be used, but to completely denaturate the RNA, e.g. for Northern blots, use 3X volumes of Formaldehyde Load Dye.

                  ii. Ethidium bromide can be added to the Formaldehyde Load Dye at a final concentration of 10 µg/ml. Some size markers may require significantly more than 10 µg/ml ethidium bromide for visualization. To accurately size your RNA, however, it is important to use the same amount of ethidium bromide in all the samples (including the size marker) because ethidium bromide concentration affects RNA migration in agarose gels.

        2) Heat denature samples at 65-70°C for 5-15 min.

        Denaturation for 5 min is typically sufficient for simply assessing RNA on a gel, but a 15 min denaturation is recommended when running RNA for a Northern blot. The longer incubation may be necessary to completely denature the RNA.

        3. Electrophoresis

        Load the gel and electrophorese at 5-6 V/cm until the bromophenol blue (the faster-migrating dye) has migrated at least 2-3 cm into the gel, or as far as 2/3 the length of the gel.

        4. Results

        Visualize the gel on a UV transilluminator. (If ethidium bromide was not added to the Formaldehyde Load Dye, the gel will have to be post-stained and destained.)

        Intact total RNA run on a denaturing gel will have sharp 28S and 18S rRNA bands (eukaryotic samples). The 28S rRNA band should be approximately twice as intense as the 18S rRNA band (Figure 1, lane 3). This 2:1 ratio (28S:18S) is a good indication that the RNA is intact. Partially degraded RNA will have a smeared appearance, will lack the sharp rRNA bands, or will not exhibit a 2:1 ratio. Completely degraded RNA will appear as a very low molecular weight smear. Inclusion of RNA size markers on the gel will allow the size of any bands or smears to be determined and will also serve as a good control to ensure the gel was run properly (Figure 1, lane 1). Note: Poly(A) selected samples will not contain strong rRNA bands and will appear as a smear from approximately 6 kb to 0.5 kb (resulting from the population of mRNAs, and depending on exposure times and conditions), with the area between 1.5 and 2 kb being the most intense (this smear is sometimes apparent in total RNA samples as well).

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