Two-dimensional and return gel electrophoresis for diagnostics of circular RNA

Denaturing conditions can be established by high concentrations of urea or of other denaturants or by high temperature. In 2D gel electrophoresis, native conditions are established in the first and fully denaturing conditions in the second dimension (Figure 7). All RNAs and DNAs from a crude extract run on or faster than a diagonal-like front (Figure 7B); because the sample is a crude extract, isolated bands are not visible. Only the circular viroid RNA is well separated as a clear band behind the front (101). In a modification of the same principle (Figure 8), all nucleic acids running faster than the viroid RNA and in front of the xylene cyanol marker have left the gel in the first dimension. The denaturing conditions were established by changing the running buffer, raising the temperature and reversing the direction of gel electrophoretic migration. The circRNAs appear to be well separated behind all other nucleic acids (102).

Two-dimensional gel electrophoresis. (A) The first electrophoresis is a conventional PAGE with native conditions. Circular (c) and linear (l) viroid RNA can not be visualized by standard staining techniques due to the high background of other nucleic acids (gray). After electrophoresis the lanes a and b are cut from the gel; these are polymerized at the bottom of a new gel matrix. (B) Due to the denaturing conditions used for the second dimension, the circular viroid, which migrates very slowly, is well separated from other nucleic acids. Modified from (101).

Return gel electrophoresis. (A) The first electrophoresis is a conventional PAGE with high-salt conditions (89 mM Tris, 89 mM boric acid, pH 8.3) and low temperature (20 °C). If the dye xylene cyanol (XC), present in the samples, is nearly running out of the gel matrix, the buffer is exchanged to low-salt conditions (10 mM Tris, 10 mM boric acid, pH 8.3, 8 M urea), the temperature is raised to 60°C and the direction of the electric field is inverted. (B) During the second electrophoresis step all nucleic acids are denatured; then the circular viroid migrates more slowly than all other, larger nucleic acids. Modified from (102).

Both methods have been applied by several research groups for diagnostic purposes (e. g. (103–105)); thus, the use of radioactively labeled or fluorescent oligonucleotides for hybridization could be completely avoided. Viroid-like, circular satellite RNAs, so-called virusoids, were diagnosed by these procedures as well (101,102). Not only 3′-5′ covalently closed circles but also 2′-5′ circles as in lariat structures were analyzed by 2D gel electrophoresis (106,107). The advantage of return gel electrophoresis is due to the ability of analyzing more samples in a single gel and to the lack or requiring repolymerization of a second gel.