RNA molecules can be analyzed on both native or denaturing agarose and polyacrylamide gels.
Non-denaturing RNA electrophoresis eliminates the need for hazardous chemicals, but due to intramolecular interactions, RNA molecules can form extensive double-stranded structures that are quite difficult to disrupt. As a result, accurate sizing of RNA molecules is not always possible under non-denaturing conditions. Native RNA electrophoresis is therefore typically used to asses the overall quality of total RNA.
Denaturing electrophoresis is recommended to precisely determine the size and integrity of RNA molecules.

Non-denaturing RNA Electrophoresis

Non-denaturing RNA electrophoresis can be performed using 50X TAE Buffer or 10X TBE Buffer and TopVision™ Agarose gels.

Denaturing RNA Electrophoresis

Types of denaturing RNA electrophoresis conditions include:

• Denaturing polyacrylamide gel electrophoresis in TBE buffer supplemented with 7 M urea. The RiboRuler™ Low Range RNA Ladder and 5% polyacrylamide-urea gel is recommended for analysis of smaller RNA molecules.
• Formaldehyde agarose gel electrophoresis in MOPS buffer. Formaldehyde forms unstable Schiff bases with the imino-groups of guanine residues. This maintains RNA in the denatured state.
• Glyoxal/DMSO agarose gel electrophoresis in sodium phosphate buffer. The two aldehyde groups of glyoxal react under slightly acid conditions with the imino-groups of guanine to form cyclic derivatives that maintain RNA in the denatured state.

Assessment of RNA Quality

After electrophoresis of total RNA samples in the presence of ethidium bromide, the 28S and 18S human rRNA should be clearly visible under UV illumination. Fast-migrating bands composed of 5.8S RNA and 5S RNA may also be visible depending on the RNA purification procedure. The intensity of the 28S RNA should be approximately twice the intensity of the 18S RNA. Smearing should not be visible around either band.
28S human rRNA migrates at approximately 5000 b while 18S human rRNA migrates at approximately 1900 b.

RNA Quantification

RiboRuler™ RNA ladders are suitable for in-gel RNA quantification. These ladders are produced from chromatography-purified RNA transcripts and are free from any degraded RNA or NTPs that often interfere with spectrophotometric measurements and lead to erroneous RNA quantification. Spectrophotometrically determined RNA concentrations of each ladder band are given in product description.
Protocols and Recommendations provides extensive information for successful RNA electrophoresis.
Troubleshooting Guide for RNA Electrophoresis provides solutions when problems arise.

Figure 1. Analysis of RiboRuler™ High Range RNA Ladder, using an Agilent 2100 bioanalyzer, RNA 6000 Nano LabChip® Kit and agarose gel electrophoresis.
A – analysis of RiboRuler™ High Range RNA Ladder on Agilent 2100 bioanalyzer
B – agarose gel analysis of RiboRuler™ High Range RNA Ladder

Products for RNA electrophoresis:

• RiboRuler™ Low Range RNA Ladder (#SM1831/3), from 100 to 1000 b
• RiboRuler™ High Range RNA Ladder (#SM1821/3), from 200 to 6000 b

These ladders are mixtures of chromotography purified RNA transcripts that form sharp bands of uniform intensity and are ideal for ssRNA sizing on both native and denaturing gels.
RiboRuler™ RNA ladders are available in ready-to-use and conventional formats. Conventional versions can be labelled radioactively and are ideal for Northern blots.

• 2X RNA Loading Dye is ideal for RNA probe and ladder preparation prior to electrophoresis. The solution contains an optimal concentration of formamide, which allows for separation of RNA molecules according to their size even under non-denaturing electrophoresis conditions. The presence of ethidium bromide in the solution allows for RNA visualization without additional staining of denaturing agarose gels.
• 50X TAE and 10X TBE buffers are free of RNases and are optimal for RNA gel electrophoresis. TAE buffer is recommended for analysis of larger RNA fragments, whereas TBE buffer is used for RNA molecules smaller than 1500 b and for denaturing polyacrylamide gel electrophoresis.
• TopVision™ Agarose and TopVision™ Low Melting Point Agarose are ribonucleases-free and are recommended for analytical and preparative RNA electrophoresis.
• GeneRuler™ and O'GeneRuler™ Ultra Low Range DNA Ladders can successfully be used for siRNA analysis (see Fig. 2 below).

Figure 2. siRNA gel analysis by GeneRuler™ and O'GeneRuler™ Ultra Low Range DNA ladders.
Electrophoresis conditions: 5% agarose gel, 1X TBE buffer, 7 V/cm, 45 min.
1 – GeneRuler™ Ultra Low Range DNA Ladder
2 – 21 bp siRNA (0.5 µg), premixed with 6X DNA Loading Dye