TABLE 1.

Comparative composition of the traditional and optimized RNA electrophoresis systems.

	Traditional system	Optimized system
Conductive media, stock concentration	MOPS/NaOAca, 10×	HTb, 50×	TTb, 50×
Composition of the stock solution	0.2M MOPS-NaOH (pH 7.0), 20 mM NaOAc, 10 mM EDTA	1.5M Hepes, 1.5M triethanolaminec	1.5M Tricine, 1.5M triethanolaminec
Running buffer (1×)	20 mM MOPS-NaOH (pH 7.0), 2 mM NaOAc, 1 mM EDTA	30 mM Hepes, 30 mM triethanolamine	30 mM Tricine, 30 mM triethanolamine
Formaldehyde concentration in gel	2.2M	0.4M
Sample composition	RNA, 50% formamide, 2.7M formaldehyde, 1×running buffer, 5% glycerol, 1 mM EDTA, 0.025% bromophenol blue, 0.025% xylene cyanol FF	RNA, 50% formamide 0.4 M formaldehyde 1×running buffer, 0.5 mM EDTA, 0.02% bromophenol blue

^aTraditional recipe, prepared according to [2].

^bpK-matched buffers, originally described by [4].

^cTo prepare 50×stock solution, start by pouring the required amount of triethanolamine in a beaker placed on a balance (note that triethanolamine is liquid). Next, add Hepes (or Tricine) and high-quality deionized water to ~0.9 of the final volume, dissolve reagents completely using a magnetic stirrer and bring to the final volume with deionized water. pH of the buffers (HT, 7.6±0.2; TT, 7.9±0.2) is established automatically and should not be adjusted. Wrong pH indicates an error in buffer preparation (e.g., using salts of Hepes and triethanolamine instead of free acid/base). With high-quality reagents, filtering freshly prepared 50×stock solutions through sterile 0.2 µm high-protein binding filters (e.g., nylon) is normally sufficient to guard against trace amounts of RNases. If desired, the stock solutions can be additionally autoclaved at 121°C for 15 min. A slight darkening that may occur after this does not affect buffer performance.