Electrophoretic Separation Of Rna A

protocol for Northern blot analysis is provided in Table 3. When analyzing total cellular RNA, the quantity of RNA loaded onto the gel can range from 5 to 30 | g. The ideal concentration should be determined empirically and depends on the quantity and quality of target mRNA as well as specificity of the probe. Analysis of purified mRNA can be performed with as little as 1.0 |g, although a greater quantity (5-10 |g) is typically used to permit multiple uses of a single membrane. Just prior to electrophoresis, the single-stranded native RNA molecules must be denatured to abolish secondary structure (see Table 2). This is accomplished by heating the samples to 65°C in the presence of formaldehyde and formamide. The denatured state is maintained during electrophoresis by the addition of formaldehyde to the agarose gel (24). Denaturation of RNA with glyoxal was introduced in 1977 as an alternative to formaldehyde denaturation (25); although glyoxal denatura-tion works well, additional steps are required to remove the glyoxal products after blotting. The standard RNA gel (1.2% agarose, 1.1% formaldehyde) allows separation of RNA from 0.5 to 6.0 kbp. It is advisable to run RNA molecular weight standards on every gel to aid in determining the molecular weight of bands present. Because RNA and DNA do not migrate at the same rate, DNA standards are not acceptable for RNA gels. RNA standards are commercially available in several size ranges. The addition of ethidium bromide to RNA gels is controversial. Although visualization of the RNA provides information on integrity and quantity, experimental evidence suggests that the subsequent transfer of RNA to nitrocellulose or nylon is impeded when ethidium bromide is present, resulting in a 12-18% decrease in final hybridization signal (13,26). For this reason, many researchers remove and stain only the lane containing the RNA standards. However, the valuable

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