rna denaturing loading dye  (New England Biolabs)

Journal: Nucleic Acids Research

Article Title: RNA denaturation underlies circular RNA separation

doi: 10.1093/nar/gkaf1160

Figure Lengend Snippet: PAGE and CGE analysis of circRNAs. ( A ) PAGE analysis of circRNAs under native and denaturing conditions. The CVB3–GFP circRNA samples (left panel) were incubated with or without RNase R. The origami RNA precursor (right panel) was incubated with or without T4 RNA ligase 2. Four different PAGE conditions were tested: (i) Native PAGE. RNA samples were mixed with native loading dye and analyzed in a 3.5% native polyacrylamide gel at 4°C (80 V for 130 min). (ii) Same as Condition 1, but run at room temperature (350 V for 16 min). (iii) Same as Condition 2 but with RNA samples pre-denatured by mixing with denaturing 2× RNA Loading Dye (NEB), heating at 90°C for 2 min, and then snap-cooling on ice before being analyzed. (iv) Same as Condition 3 but using denaturing PAGE (7.5 M urea). ( B ) Denaturing PAGE analysis of circRNAs in gels at indicated acrylamide concentrations. Pre-denatured RNA samples were analyzed by 7.5 M urea PAGE at 350 V for 20 or 30 min at room temperature. ( C ) CGE of circRNAs. Products from the denoted RNA constructs were analyzed using the 2100 Bioanalyzer (Agilent). For the pre-denaturing condition (right), RNAs were mixed with 2× denaturing RNA loading dye, heated at 65°C for 2 min, and then snap-cooled on ice prior to chip loading. The gel bands are annotated with symbols as defined in Fig. .

Article Snippet: RNA samples were mixed with a denaturing 2× RNA Loading Dye (95% formamide, 0.02% SDS, 0.02% bromophenol blue, 0.01% xylene cyanol, 1 mM EDTA; NEB), followed by snap cooling (90°C for 1 min, then on ice for at least 5 min).

Techniques: Incubation, Clear Native PAGE, Construct