Size-fractionation of RNA by hot agarose electrophoresis.

Since RNA molecules commonly form stable secondary structures, agarose gel size-fractionation of RNA is routinely achieved by chemical denaturing conditions during electrophoresis. This implies the use of toxic compounds such as methylmercuric hydroxide (1), formaldehyde (3) or cumbersome electrophoretic conditions using glyoxal and dimethylsulfoxide (4) after RNA denaturation. Here, we describe an alternative method in which the RNA molecules are preserved in a denatured state simply by maintaining a high temperature (ca. 50°C) during the electrophoretic separation. To prove the feasibility of the proposed system, we have focused our analysis on ribosomal RNA (rRNA) molecules because it is well known that they form strong, stable secondary structures. We have compared the relative electrophoretic mobility of rRNAs from three distantly related organisms (rat, fruit fly and bacteria) using three different systems: standard non-denaturing agarose gel electrophoresis (5) in 1× TAE buffer (40 mM Tris-acetate, pH 8.0, 1 mM EDTA), formaldehyde agarose gel electrophoresis (2) in HEPES buffer (20 mM HEPES, pH 7.4, 1 mM EDTA, pH 8.0, 0.45 M formaldehyde) and the hot-gel system described below. Moreover, we also demonstrated that RNA size-fractionated under hot conditions is perfectly suitable for blotting and hybridization analysis with a nonradioactive DNA probe, and it gives higher sensitivity in the Northern blot than other optimized procedures that use formaldehyde gels. Total RNA was extracted employing TRI REAGENT (Sigma, St. Louis, MO, USA) following the manufacturer’s instructions. For both hot and formaldehyde gel electrophoresis, 10 μL of total RNA preparations were mixed with 10 μL of formaldehyde buffer (40 mM HEPES, pH 7.4, 2 mM EDTA, pH 8.0, 4 M formaldehyde, 60% formamide), denatured by heating at 75°C for 5 min and chilled immediately on ice/ethanol. Then, 2 μL of gel loading buffer (50% glycerol, 1 mM EDTA pH 8.0, 0.25% bromophenol blue, 0.25% xylene cyanol) were added to the samples. For non-denaturing agarose gel electrophoresis, 1 μL of loading buffer was added to 10 μL of RNA samples. In all cases, the electrophoresis was run in 1.2% agarose gels using the GNA 100 horizontal gel apparatus (Amersham Pharmacia Biotech, Uppsala, Sweden). Separations were ended when the bromophenol dye marker reached the bottom of the gel slab (45 min for the hot agarose and 2 h for the formaldehyde gel). To achieve the hot conditions during electrophoresis, the 1× TAE buffer was heated to 60°C, poured into the apparatus in which the gel had already been placed and, when the temperature reached 50°C, a 15 min pre-electrophoresis at 10 V/cm (130–140 mA) was conducted. Subsequently, denatured RNA samples were loaded and the gel was run at 5 V/cm. Under these conditions, the buffer temperature remained constant at 50°C ± 2°C. Gels were stained in 0.5 μg/mL ethidium bromide in diethylpyrocarbonate (DEPC)-treated water for 15 min and visualized under UV light. In the case of the formaldehyde gel, satisfactory visualization of bands required two subsequent 30 min destaining washes in DEPC-treated water. For Northern analysis, we carried out nonradioactive detection of rat glyceraldehyde-3-phosphate dehydrogenase RNA. A 400 bp DNA fragment was labelled with DIG-dUTP using the random primed labeling kit (Roche Molecular Biochemicals, Mannheim, Germany). In the case of the formaldehyde gel, downward transfer was carried out according to the improved method of Ingelbrecht et al. (2). In the case of hot agarose gel electrophoresis, the transfer was accomplished by standard downward capillary using 10× standard saline citrate (SSC) as transfer buffer (20× SSC is 3 M NaCl, 0.3 M sodium citrate, pH 7.0). In both cases, the transfers were stopped after 2.5 h, membranes were dried for 15 min at 37°C and fixed for 1.5 min under UV. Both membranes were prehybridized together in 25 mL hybridization solution containing 50% formamide, 5× SSC, Benchmarks